Sample plate systems and methods

ABSTRACT

A sample plate comprising a sample well is disclosed. The sample well can comprise one or more bead retaining chambers. A method of using the sample plate and a kit comprising the sample plate is also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.13/187,791, pending, filed on 21 Jul. 2011 which claims the benefit ofUnited Kingdom Patent Application Nos. GB 1101222.6, filed on Jan. 25,2011 and GB 1106618.0, filed on Apr. 19, 2011, each of which isincorporated herein by reference in its entirety. This application is acontinuation-in-part application of U.S. Ser. No. 12/846,580, filed onJul. 29, 2010, now U.S. Pat. No. 8,541,246, which claims priority toUnited Kingdom Patent Application Nos. GB 0913258.0, filed on Jul. 29,2009; GB 0917555.5 filed on Oct. 7, 2009; and GB 1006087.9 filed on Apr.13, 2010, each of which is incorporated herein by reference in itsentirety. This application is also related to PCT Patent Application No.PCT/GB2010/001443, filed on Jul. 29, 2010, which is incorporated hereinby reference in its entirety.

BACKGROUND

The present invention relates to a sample plate, an automated apparatus,a reagent bead or microsphere dispenser, a method of dispensing reagentbeads or microspheres, a kit for performing Enzyme Linked ImmunosorbentAssay procedures, a kit for performing nucleic acid probe procedures, amethod of manufacturing a sample plate and a computer program executableby the control system of an automated apparatus.

An automated reagent bead or microsphere dispenser for dispensingreagent beads or microspheres into a sample plate is disclosed. Thesample plate may be used to carry out diagnostic testing such as EnzymeLinked Immunosorbent Assay (“ELISA”) procedures or other immunoassayprocedures. Alternatively, the sample plate may be used to carry outtesting for DNA or RNA sequences.

In one embodiment, immunoassay procedures are used to test biologicalproducts. These procedures can exploit the ability of antibodiesproduced by the body to recognize and combine with specific antigenswhich may, for example, be associated with foreign bodies such asbacteria or viruses, or with other body products such as hormones. Oncea specific antigen-antibody combination has occurred it can be detectedusing chromogenic, fluorescent or chemiluminescent materials or lesspreferably by using radioactive substances. Radioactive substances areless preferred due to environmental and safety concerns regarding theirhandling, storage and disposal. The same principles can be used todetect or determine any materials which can form specific binding pairs,for example using lectins, rheumatoid factor, protein A or nucleic acidsas one of the binding partners.

In one embodiment, ELISA is a form of immunoassay procedure used,wherein one member of the binding pair is linked to an insoluble carriersurface (“the solid phase”) such as a sample vessel, and after reactionthe bound pair is detected by use of a further specific binding agentconjugated to an enzyme (“the conjugate”). The characteristics andchoice of solid phases for capture assays, on methods and reagents forcoating solid phases with capture components, on the nature and choiceof labels, and on methods for labeling components is well known in thearts and may also be applied to assays for other specific binding pairs.An example of a standard textbook is “ELISA and Other Solid PhaseImmunoassays, Theoretical and Practical Aspects”, Editors D. M. Kemeny &S. J. Challacombe, published by John Wiley, 1988. Such advice.

In one embodiment of ELISA, the solid phase is coated with a member ofthe binding pair. An aliquot of the specimen to be examined canincubated with the solid coated solid phase and any analyte that may bepresent is captured onto the solid phase. After washing to removeresidual specimen and any interfering materials it may contain, a secondbinding agent, specific for the analyte and conjugated to an enzyme canbe added to the solid phase. During a second incubation any analytecaptured onto the solid phase can combine with the conjugate. After asecond washing to remove any unbound conjugate, a chromogenic substratefor the enzyme can be added to the solid phase. Any enzyme present canbegin converting the substrate to a chromophoric product. After aspecified time the amount of product formed may be measured using aspectrophotometer, either directly or after stopping the reaction.

Many variants are known in the art including fluorogenic and luminogenicsubstrates for ELISA, direct labeling of the second member of thebinding pair with a fluorescent or luminescent molecule (in which casethe procedure is not called an ELISA but the process steps are verysimilar) and nucleic acids or other specific pairing agents instead ofantibodies as the binding agent. In some embodiments, the assays usefluid samples, e.g. blood, serum, urine, etc., which are aspirated froma sample tube and are then dispensed into a solid phase. Samples may bediluted prior to being dispensed into the solid phase or they may bedispensed into deep well microplates, diluted in situ and then thediluted analyte may be transferred to the functional solid phase.

In one embodiment, the solid phase is a standard sample vessel known asa microplate, which can be stored easily and which may be used with avariety of biological specimens. The microplate can be made frommaterials including, but not limited to polystyrene, PVC, Perspex orLucite. In one embodiment, the microplate measures approximately 5inches (12.7 cm) in length, 3.3 inches (8.5 cm) in width, and 0.55inches (1.4 cm) in depth. In one embodiment, the microplate is made frompolystyrene wherein the polystyrene's enhanced optical clarity assistsvisual interpretation of the results of a reaction. The polystyrenemicroplate can also be compact, lightweight and easily washable. In oneembodiment, the microplate is sold under the name “MICROTITRE”®. Themicroplate can comprise 96 wells (also known as “microwells”) which canbe symmetrically arranged in an 8×12 array. The microwells can have amaximum volume capacity of approximately 350 μl. In one embodiment,approximately 10-200 μl of fluid is dispensed into a microwell. In somearrangements of the microplate the microwells may be arranged in stripsof 8 or 12 wells that can be moved and combined in a carrier to give acomplete plate having conventional dimensions.

Positive and negative controls can be supplied with microplates, such aswith commercial kits, and are used for quality control and to provide arelative cut-off. After reading the processed microplate, the results ofthe controls can be checked against the manufacturer's validated valuesto ensure that the analysis has operated correctly and then the value isused to distinguish positive from negative specimens and a cut off valueis calculated. Standards can be provided for quantitative assays andused to build a standard curve from which the concentration of analytein a specimen may be interpolated.

In one embodiment, the ELISA procedure can involve multiple stepsincluding, but not limited to, one or more of the following: pipetting,incubation, washing, transferring microplates between activities,reading and data analysis. One or more of the steps (or “phases”)involved in the ELISA procedures such as sample distribution, dilution,incubation at specific temperatures, washing, enzyme conjugate addition,reagent addition, reaction stopping and the analysis of results, can beautomated. For example, the pipette mechanism used to aspirate anddispense fluid samples uses disposable tips which are ejected afterbeing used so as to prevent cross-contamination of patients' samples.Multiple instrumental controls can be in place to ensure thatappropriate volumes, times, wavelengths and temperatures are employed,data transfer and analysis is fully validated and monitored. Automatedimmunoassay apparatus for carrying out ELISA procedures can be used inlaboratories of e.g. pharmaceutical companies, veterinary and botanicallaboratories, hospitals and universities for in-vitro diagnosticapplications such as testing for diseases and infection, and forassisting in the production of new vaccines and drugs.

ELISA kits can comprise microplates having microwells which have beencoated with a specific antibody (or antigen). For example, in the caseof a hepatitis B antigen diagnostic kit, the kit manufacturer willdispense anti-hepatitis B antibodies which have been suspended in afluid into the microwells of a microplate. The microplate is thenincubated for a period of time, during which time the antibodies adhereto the walls of the microwells up to the fluid fill level (typicallyabout half the maximum fluid capacity of the microwell). The microwellsare then washed leaving a microplate having microwells whose walls areuniformly covered with anti-hepatitis B antibodies up to the fluid filllevel.

A testing laboratory can receive a number of sample tubes containing,for example, body fluid from a number of patients. A specified amount offluid can be aspirated out of the sample tube using a pipette mechanismand dispensed into one or more microwells of a microplate that has beenpreviously prepared by the manufacturer, such as discussed above. If itis desired to test a patient for a number of different diseases, fluidfrom the patient is typically dispensed into a number of separatemicroplates, wherein each microplate may have been coated by itsmanufacturer with a different binding agent. Each microplate can then beprocessed separately to detect the presence of a different disease. Thiscan lead to analysis of several different analytes with a multiplicityof microplates and transfer of aliquots of the same specimen to thedifferent microplates, resulting in large numbers of processing stepsand incubators and washing stations that can cope with many microplatesvirtually simultaneously. In automated systems this instruments may havemultiple incubators and complex programming to avoid clashes betweenmicroplates with different requirements. For manual operation eitherseveral technicians may be needed or the throughput of specimens isslow. It is possible to combine strips of differently coated microwellsinto a single carrier, add aliquots of a single specimen to thedifferent types of well and then perform the ELISA in this combinedmicroplate. Constraints on assay development, however, can make thiscombination difficult to achieve and can lead to errors of assignment ofresult, while manufacture of microplates with several different coatingsin different microwells can present difficulties of quality control.

Conventional ELISA techniques have typically concentrated uponperforming the same single test upon a plurality of patient samples permicroplate or in detecting the presence of one or more of a multiplicityof analytes in those patients without distinguishing which of thepossible analytes is actually present. For example, it is commonplace todetermine in a single microwell whether a patient has antibodies to HIV1 or HIV 2, or HIV 1 or 2 antigens, without determining which analyte ispresent and similarly for HCV antibodies and antigens.

However, a new generation of assays are being developed which enablemultiplexing to be performed. Multiplexing enables multiple differenttests to be performed simultaneously upon the same patient sample.

In one embodiment, multiplexing provides a microplate comprising 96sample wells wherein an array of different capture antibodies isdisposed in each sample well. The array can comprise an array of 20 nLspots each having a diameter of 350 μm. The spots can be arranged with apitch spacing of 650 μm. Each spot corresponds with a different captureantibody.

Multiplexing enables a greater number of data points and moreinformation per assay to be obtained compared with conventional ELISAtechniques wherein each sample plate tests for a single analyte ofinterest. The ability to be able to combine multiple separate tests intothe same assay can lead to considerable time and cost savings.Multiplexing also enables the overall footprint of the automatedapparatus to be reduced.

Provided herein is a sample plate and associated automated apparatuswhich has an improved format and which provides a greater flexibility.

In addition to ELISA procedures, a hybridization probe can be used totest for the presence of DNA or RNA sequences. A hybridization probetypically comprises a fragment of nucleic acid, such as DNA or RNA,which is used to detect the presence of nucleotide sequences which arecomplementary to the nucleic acid sequence of the probe. Thehybridization probe can hybridize to single-stranded nucleic acid (e.g.DNA or RNA) whose base sequence allows pairing due to complementaritybetween the hybridization probe and the sample being analyzed. Thehybridization probe may be tagged or labeled with a molecular markersuch as a radioactive or more preferably a fluorescent molecule. Theprobes are inactive until hybridization at which point there is aconformational change and the molecule complex becomes active and willthen fluoresce (which can be detected under UV light) DNA sequences orRNA transcripts which have a moderate to high sequence similarity to theprobe are then detected by visualizing the probe under UV light.

It is desired to provide an improved sample plate for retaining reagentbeads, as well as related systems and methods.

SUMMARY

Disclosed herein are sample plates comprising one or more sample wells,wherein one or more of the sample wells comprise: (a) a base portion;and (b) one or more recesses provided in the base portion; wherein eachof the one or more recesses has a dimension for a bead deposited in thewell to be substantially retained or secured within the recess, and thebead forms a substantially fluid-tight circumferential seal with a wallof the base portion which defines the recess. In one embodiment, the oneor more recesses comprise a blind recess or an open through hole. In oneembodiment, the recess is substantially cylindrical. In one embodiment,an opening to the recess is circular. In one embodiment, the recess isconical and has a first diameter which is greater than a diameter of abead deposited in the recess and a second diameter which is less than adiameter of the bead deposited in the recess. In one embodiment, thethrough recess has a taper selected from the group consisting of: (i)<0.5°; (ii) 0.5°; (iii) 0.5-1°; (iv) 1-2°; (v) 2-4°; (vi) 4-6°; (vii)6-8°; (viii) 8-10°; and (ix) >10°. In one embodiment, the diameter ordepth of the recess is selected from the group consisting of: (i) <0.5mm; (ii) 0.5-1.0 mm; (iii) 1.0-1.5 mm; (iv) 1.5-2.0 mm; (v) 2.0-2.5 mm;(vi) 2.5-3.0 mm; (vii) 3.0-3.5 mm; (viii) 3.5-4.0 mm; (ix) 4.0-4.5 mm;(x) 4.5-5.0 mm; (xi) <5.0 mm; and (xii) >5.0 mm. In one embodiment, inat least one sample well the base portion is segmented into a pluralityof segments which are arranged at different heights relative to eachother. In one embodiment, in at least one sample well, the sample wellfurther comprises one or more baffles or dividers which separates ordivides the base portion into at least a first region and a secondregion. In one embodiment, the one or more baffles or dividers attenuateor eliminate light reflected off one or more reagent beads located inthe first region from impinging upon one or more reagent beads locatedin the second region. In one embodiment, the one or more recessescomprise a countersunk or enlarged portion for facilitating theinsertion of a bead into one or more of the through holes or recesses.In one embodiment, the one or more sample wells comprise between 2 and22 recesses. In one embodiment, the recesses are arrangedcircumferentially around a central portion of the sample well. In oneembodiment, the central portion comprises a central recess. In oneembodiment, the central portion does not comprise a recess. In oneembodiment, the plurality of recesses is arranged in a substantiallysymmetrical or regular manner. In one embodiment, the plurality ofrecesses is arranged in a substantially asymmetrical or irregularmanner. In one embodiment, the plurality of recesses is arranged in asubstantially linear manner. In one embodiment, the plurality ofrecesses is arranged in a substantially curved manner. In oneembodiment, the sample plate comprises sample wells arranged in an A×Bformat, wherein A and B are perpendicular axes, and the number of wellsalong the A axis can be greater than, less than, or equal to the numberof wells along the B axis. In one embodiment, the number of wells alongthe A axis or B axis is at least 2. In one embodiment, the number ofwells along the A axis or B axis is between 2 and 15. In one embodiment,one of the sample wells is connected to another sample well by afrangible region. In one embodiment, the sample plate comprises a basecomprising a docking portion for securing the sample plate to acorresponding docking portion of a plate frame holder. In oneembodiment, the sample plate of claim further comprises a bead. In oneembodiment, the bead is attached to a probe. In one embodiment, theprobe is a nucleic acid, antibody, antibody fragment, protein, peptide,aptamer, or chemical compound.

Also disclosed herein are bead dispensing systems comprising: (a) a beaddispenser; (b) a sample plate comprising a sample well, wherein thesample well comprises a base portion, wherein the base portion comprisesone or more recesses, wherein each of the one or more recesses has adimension for a bead deposited in the well to be substantially retainedor secured within the recess, and the bead forms a substantiallyfluid-tight circumferential seal with a wall of the base portion whichdefines the recess; and (c) a control system configured to controldispensing of the bead from the bead dispenser into the sample plate. Inone embodiment, the one or more recesses comprise a blind recess or anopen through hole. In one embodiment, the bead dispenser comprises: (i)a syringe body comprising an annular chamber surrounding a longitudinalbore, wherein the annular chamber is configured to channel a reagentbead within the annular chamber towards a chamber provided in the bore;(ii) a plunger provided within the longitudinal bore; and (iii) a barrelor nozzle; wherein the plunger is configured to dispense a bead from thechamber into the barrel or nozzle. In one embodiment, the bead dispenseris configured to dispense a plurality of beads automatically.

Also disclosed herein are methods of dispensing beads comprising: (a)providing a bead dispenser comprising a bead; (b) providing a sampleplate comprising a sample well, wherein the sample well comprises a baseportion; wherein the base portion comprises one or more recesses,wherein each of the one or more recesses has a dimension for a beaddeposited in the well to be substantially retained or secured within therecess, and the bead forms a substantially fluid-tight circumferentialseal with a wall of the base portion which defines the recess; and (c)controlling the dispensing of the bead from the bead dispenser into thesample plate. In one embodiment, the one or more recesses comprise ablind recess or an open through hole. In one embodiment, the dispensingis performed automatically.

Also disclosed herein are kits for detecting an analyte comprising: (a)a plurality of beads; and (b) sample plate comprising a sample well,wherein the sample well comprises a base portion; wherein the baseportion comprises one or more recesses, wherein each of the one or morerecesses has a dimension for a bead deposited in the well to besubstantially retained or secured within the recess, and the bead formsa substantially fluid-tight circumferential seal with a wall of the baseportion which defines the recess. In one embodiment, the one or morerecesses comprise a blind recess or an open through hole. In oneembodiment, the plurality of beads comprises one or more probes. In oneembodiment, the probe is a nucleic acid, antibody, antibody fragment,protein, peptide, aptamer, or chemical compound.

Also disclosed herein are methods of detecting an analyte comprising:(a) adding a sample to a sample plate comprising a sample well, whereinthe sample well comprises a base portion; wherein the base portioncomprises one or more recesses, wherein each of the one or more recesseshas a dimension for a bead deposited in the well to be substantiallyretained or secured within the recess, and the bead forms asubstantially fluid-tight circumferential seal with a wall of the baseportion which defines the recess; and (b) detecting binding of ananalyte in the sample with the probe. In one embodiment, the recesscomprises a blind recess and an open through hole. In one embodiment,the sample plate comprises a plurality of probes and a plurality ofanalytes are detected. In one embodiment, a plurality of samples isadded to the sample plate.

Also disclosed herein are methods of manufacturing a sample platecomprising: (a) providing a sample plate comprising one or more samplewells each having a base portion; and (b) forming one or more recessesin the one or more base portions, wherein each of the one or morerecesses has a dimension for a bead deposited in the well to besubstantially retained or secured within the recess, and the bead formsa substantially fluid-tight circumferential seal with a wall of the baseportion which defines the recess. In one embodiment, the one or morerecesses comprises a blind recess or an open through hole.

According to an aspect of the present invention there is provided asample plate comprising one or more sample wells, wherein one or more ofthe sample wells comprise:

a base portion; and

one or more open through holes provided in the base portion;

characterized in that:

a reagent bead or microsphere is substantially retained or secured, inuse, within the through hole so as to form a substantially fluid-tightcircumferential seal with a wall of the base portion which defines thethrough hole.

The one or more through holes pass from the bottom of the sample well tothe rear or bottom surface of the sample plate. As a result, if areagent bead is not retained or secured within the open through holethen any fluid in the sample well can leak out of the sample well viathe through hole.

According to an aspect of the present invention there is provided asample plate comprising one or more sample wells, wherein one or more ofthe sample wells comprise:

a base portion; and

one or more recesses provided in the base portion;

characterized in that:

a reagent bead or microsphere is substantially retained or secured, inuse, within the recess so as to form a substantially fluid-tightcircumferential seal with a wall of the base portion which defines therecess.

It should be understood that a circular bead within a hole, bore orrecess having a square cross-section will not form a fluid-tightcircumferential seal with the wall defining the hole, bore or recess. Afluid-tight circumferential seal should be understood as meaning that abarrier is formed around the entire circumference of the bead and thewall defining the hole, bore or recess.

In some embodiments reagent beads or microspheres are retained orsecured within a through hole or a recess formed in the base portion ofthe sample plate. Each reagent bead or microsphere forms a fluid-tightand/or water-tight and/or air-tight seal about the entire outer diameteror circumference of the reagent bead or microsphere.

Once the reagent bead or microsphere is located within the through holeor recess, then fluid is substantially prevented from being able to passfrom one side of the through hole or recess to the other side by thereagent bead or microsphere which forms a tight seal about the entirecircumference of the reagent bead or microsphere.

Various different embodiments are contemplated.

In some embodiments, the sample plate comprises one or more recesses,wherein the one or more recesses preferably comprise blind recesses thatare closed at one end. A blind recess differs from a through hole inthat if a reagent bead is not retained or secured in a blind recess thensample fluid within a sample well will not leak out of the sample well.

In some embodiments, open through holes or recesses provided in the baseportion of a sample well can be substantially cylindrical and have adiameter less than a diameter of a reagent bead or microsphere depositedin the through hole or the recess so that the reagent bead ormicrosphere is retained or secured within the through hole or within therecess by an interference or friction fit.

The open through hole or the recess can, according to anotherembodiment, be conical and have a first diameter which is greater than adiameter of a reagent bead or microsphere deposited in the through holeor in the recess and a second diameter which is less than a diameter ofthe reagent bead or microsphere deposited in the through hole or in therecess. In some embodiments, reagent beads or microspheres can besecured within the through hole by the taper.

The first diameter can be distal from a portion of the base portion ontowhich sample fluid is dispensed, in use, and the second diameter wouldbe proximate to the portion of the base portion onto which sample fluidis dispensed in use.

Alternatively, the first diameter can be proximate to a portion of thebase portion onto which sample fluid is dispensed, in use, and thesecond diameter would be distal from the portion of the base portiononto which sample fluid is dispensed in use.

The through hole or the recess can have a taper selected from the groupconsisting of: (i) <0.5°; (ii) 0.5°; (iii) 0.5-1°; (iv) 1-2°; (v) 2-4°;(vi) 4-6°; (vii) 6-8°; (viii) 8-10°; and (ix) >10°.

An opening to the through hole or recess is preferably circular.

The through hole or recess preferably has a circular cross-sectionalshape or profile. According to an embodiment, the through holes orrecesses may have a circular cross-section along at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% or 100% of the length or depth of the through hole or recess.

In some embodiments the diameter of the through hole or the recess isselected from the group consisting of: (i) <0.5 mm; (ii) 0.5-1.0 mm;(iii) 1.0-1.5 mm; (iv) 1.5-2.0 mm; (v) 2.0-2.5 mm; (vi) 2.5-3.0 mm;(vii) 3.0-3.5 mm; (viii) 3.5-4.0 mm; (ix) 4.0-4.5 mm; (x) 4.5-5.0 mm;(xi) <5.0 mm; and (xii) >5.0 mm.

In some embodiments, the depth of the through hole or the recess isselected from the group consisting of: (i) <0.5 mm; (ii) 0.5-1.0 mm;(iii) 1.0-1.5 mm; (iv) 1.5-2.0 mm; (v) 2.0-2.5 mm; (vi) 2.5-3.0 mm;(vii) 3.0-3.5 mm; (viii) 3.5-4.0 mm; (ix) 4.0-4.5 mm; (x) 4.5-5.0 mm;(xi) <5.0 mm; and (xii) >5.0 mm.

According to an embodiment in at least one sample well (or in all thesample wells) the base portion preferably comprises a plurality of openthrough holes and/or a plurality of recesses and wherein at least some(or all) of the plurality of open through holes and/or at least some (orall) of the plurality of recesses are arranged so that there is nodirect line of sight between reagent beads retained or secured inadjacent open through holes and/or so that there is no direct line ofsight between reagent beads retained or secured in adjacent recesses.

In some embodiments, at least one sample well (or in all the samplewells) the base portion can comprise a plurality of open through holesand/or a plurality of recesses and wherein the base portion is segmentedinto a plurality of segments which are arranged at different heightsrelative to each other.

In some embodiments, at least one sample well (or in all the samplewells) the base portion can comprise a plurality of open through holesand/or a plurality of recesses and wherein the sample well furthercomprises one or more baffles or dividers which preferably separates ordivides the base portion into at least a first region and a secondregion.

The one or more baffles or dividers can be arranged so as: (i) toattenuate or eliminate light reflected off one or more reagent beadslocated in the first region from impinging upon one or more reagentbeads located in the second region; and/or (ii) to attenuate oreliminate light reflected off one or more reagent beads located in thesecond region from impinging upon one or more reagent beads located inthe region.

In some embodiments, one or more open through holes or recesses cancomprise a countersunk or enlarged portion for facilitating theinsertion of a reagent bead or microsphere into one or more of thethrough holes or recesses.

The one or more sample wells can comprise a plurality of through holesor recesses, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 or 21 through holes or recesses which areeach arranged and adapted to receive, in use, a reagent bead ormicrosphere.

The one or more through holes or recesses provided in the base portioncan be arranged:

(i) circumferentially around a central portion of the sample well; or

(ii) with a plurality of through holes or recesses arrangedcircumferentially around a central through hole or recess; or

(iii) in a substantially close-packed manner; or

(iv) in a substantially symmetrical or asymmetrical manner; or

(v) in a substantially linear or curved manner; or

(vi) in a substantially regular or irregular manner; or

(vii) in an array; or

(viii) in a circle or two or more concentric circles with no throughhole or recess located at the centre of the base portion.

In some embodiments, the sample plate comprises sample wells arranged inan A×B format wherein:

A is selected from the group consisting of: (i) 1; (ii) 2; (iii) 3; (iv)4; (v) 5; (vi) 6; (vii) 7; (viii) 8; (ix) 9; (x) 10; and (xi) >10; and

B is selected from the group consisting of: (i) 1; (ii) 2; (iii) 3; (iv)4; (v) 5; (vi) 6; (vii) 7; (viii) 8; (ix) 9; (x) 10; and (xi) >10.

One or more of the sample wells can be interconnected to one or moreother sample wells by one or more frangible regions or connections sothat the sample plate can be separated by a user into a plurality ofsmaller sample plates, sample strips or individual sample wells.

In some embodiments, the sample plate is an Immunoassay sample plate.

The sample plate can, in some embodiments, comprise a hybridizationprobe for detecting the presence of complementary DNA or RNA samples.

The sample plate can comprise a base having a female, male or otherdocking portion for securing the sample plate to a corresponding male,female or other docking portion of a plate frame holder.

Disclosed herein are combinations of a sample plate as described aboveand one or more reagent beads or microspheres inserted or located in oneor more of the through holes or recesses of the one or more samplewells.

At least some or substantially all of the reagent beads or microspherescan carry, comprise or are otherwise coated with a reagent, wherein thereagent is arranged and adapted to assay for an analyte of interest in asample liquid.

At least some or substantially all of the reagent beads or microspherescan carry, comprise or are otherwise coated with a nucleic acid probe,wherein the nucleic acid probe is arranged and adapted to hybridize withsingle-stranded nucleic acid, DNA or RNA.

Also disclosed herein are combinations of a plate frame holder and asample plate as described above.

In some embodiments, the plate frame holder can comprise a male, femaleor other docking portion for firmly securing the sample plate to theplate frame holder.

Also disclosed herein are automated apparatuses comprising:

one or more reagent bead or microsphere dispensers;

a sample plate as described above; and

a control system arranged and adapted to control the dispensing ofreagent beads or microspheres from the one or more reagent bead ormicrosphere dispensers into one or more sample wells of the sampleplate.

The one or more reagent bead or microsphere dispensers can comprise:

a syringe body comprising an annular chamber surrounding a longitudinalbore, wherein the annular chamber is arranged, in use, to channel orfunnel reagent beads or microspheres provided within the annular chambertowards a chamber provided in the bore;

a plunger provided within the longitudinal bore; and

a barrel or nozzle;

wherein the plunger is arranged, in use, to dispense a reagent bead ormicrosphere from the chamber into the barrel or nozzle.

Also provided herein are apparatuses for assaying a liquid for one ormore analytes of interest, the apparatus comprising:

one or more reagent bead or microsphere dispensers; and

a sample plate as described above.

Also disclosed herein are methods comprising:

providing a sample plate comprising one or more sample wells, whereinone or more of the sample wells comprise a base portion and one or moreopen through holes provided in the base portion; and

retaining or securing a reagent bead or microsphere within a throughhole so as to form a substantially fluid-tight circumferential seal witha wall of the base portion which defines the through hole.

Also provided herein are methods comprising:

providing a sample plate comprising one or more sample wells, whereinone or more of the sample wells comprise a base portion and one or morerecesses provided in the base portion; and

retaining or securing a reagent bead or microsphere within a recess soas to form a substantially fluid-tight circumferential seal with a wallof the base portion which defines the recess.

Also provided herein are methods comprising:

providing one or more reagent bead or microsphere dispensers;

providing a sample plate as described above; and

controlling the dispensing of reagent beads or microspheres from the oneor more reagent bead or microsphere dispensers into one or more of thesample wells.

Disclosed herein are methods of using a sample plate to analyze a samplefor multiple analytes comprising:

providing a sample plate as described above;

optionally inserting one or more reagent beads or microspheres into oneor more through holes or recesses of a sample well; and

adding a sample to the sample well.

The reagent beads or microspheres can be inserted into one or more ofthe pockets, recesses or bores of the sample wells either by the sampleplate manufacturer of by the end user.

Disclosed herein are methods of using an Enzyme Linked ImmunosorbentAssay (ELISA) to detect an antigen or an antibody in a samplecomprising:

providing a sample plate as described above;

optionally inserting one or more reagent beads or microspheres into oneor more through holes or recesses of a sample well; and

adding a sample to the sample well.

The reagent beads or microspheres can be inserted into one or more ofthe pockets, recesses or bores of the sample wells either by the sampleplate manufacturer of by the end user.

Also provided herein are methods of using a nucleic acid probe to detecta DNA or RNA sequence in a sample comprising:

providing a sample plate as described above;

optionally inserting one or more reagent beads or microspheres into oneor more through holes or recesses of a sample well; and

adding a sample to the sample well.

The reagent beads or microspheres can be inserted into one or more ofthe pockets, recesses or bores of the sample wells either by the sampleplate manufacturer of by the end user.

Disclosed herein are methods for assaying for one or more analytes ofinterest in a sample comprising:

optionally inserting one or more reagent beads or microspheres into oneor more through holes or recesses of one or more sample wells of asample plate so as to retain or secure a reagent bead or microspherewithin the through hole or recess so as to form a substantiallyfluid-tight circumferential seal with a wall of the base portion whichdefines the through hole or recess.

The reagent beads or microspheres can be inserted into one or more ofthe pockets, recesses or bores of the sample wells either by the sampleplate manufacturer of by the end user.

Disclosed herein are methods of detecting an analyte comprising:

providing a sample plate as described above wherein one or more reagentbeads or microspheres are retained or secured within one or more throughholes or recesses provided in the base portion of the sample plate;

adding a sample to the sample plate; and

detecting binding of an analyte in the sample to a reagent bead ormicrosphere.

Some embodiments further comprise one or more of the following steps:

(i) incubating the sample plate; and/or

(ii) washing the sample plate; and/or

(iii) aspirating the sample plate; and/or

(iv) adding an enzyme conjugate to the sample plate; and/or

(v) adding a visualizing agent to the sample plate; and/or

(vi) visually analyzing the sample plate.

Provided herein are kits for performing an Enzyme Linked ImmunosorbentAssay (ELISA) procedure comprising:

one or more sample plates as described above; and

a plurality of reagent beads or microspheres, the reagent beads ormicrospheres being coated with a reagent comprising an antibody, anantigen or another biomolecule.

Also provided herein are kits for performing a nucleic acid probeprocedure comprising: one or more sample plates as described above; and

a plurality of reagent beads or microspheres, the reagent beads ormicrospheres being coated with a DNA or RNA sequence.

One or more reagent beads or microspheres can be retained or securedwithin one or more through holes or recesses provided in the baseportion of the sample plate.

According to an aspect of the disclosure there are provided kits fordetecting an analyte comprising:

one or more sample plates as described above; and

a plurality of reagent beads or microspheres retained or secured withinone or more through holes or recesses provided in the base portion ofthe sample plate so that the plurality of reagent beads or microspheresform a substantially fluid-tight circumferential seal with a wall of thebase portion which defines the through hole or the recess.

Disclosed herein are methods of manufacturing a sample plate comprising:

providing a sample plate comprising one or more sample wells each havinga base portion; and

forming one or more through holes or recesses in the one or more baseportions, wherein the one or more through holes or recesses are arrangedand adapted so as to retain or secure a reagent bead or microspherewithin the through hole or recess so as to form a substantiallyfluid-tight circumferential seal with a wall of the base portion whichdefines the through hole or recess.

In some embodiments, the method of manufacturing can further compriseinserting one or more reagent beads or microspheres into the throughholes or recesses so that the one or more reagent beads or microspheresform a substantially fluid-tight circumferential seal with a wall of thebase portion which defines the through hole or recess.

Also provided herein are sample plates comprising a sample well, whereinthe sample well comprises a base portion, wherein the base portioncomprises a recess and the recess comprises a diameter less than adiameter of a bead deposited in the sample well.

The diameter of the recess is can at least about 5% smaller than thediameter of the bead.

The recess can comprise a countersunk portion.

The sample plate can comprise a plurality of sample wells.

The sample well can comprise a plurality of recesses.

The one or more recesses can comprise circular recesses.

The one or more recesses can have a circular cross-sectional shape orprofile.

According to an embodiment, a bead is substantially retained or secured,in use, within the one or more recesses by an interference or frictionfit with the recess or bore or with the circumference of the recess orbore.

According to an embodiment, a preset force can compress a bead and/ordeform the recess so as to create or enhance an interference or frictionfit with the recess or bore.

According to an embodiment, a bead forms a substantially fluid-tightseal with the recess.

In some embodiments, the one or more recesses do not comprise a taperedsection.

The sample well can comprise between 2 and 20 recesses.

According to an embodiment the sample well comprises at least 10recesses.

The plurality of recesses can be arranged circumferentially around acentral portion of the sample well.

In some embodiments the central portion can comprise a central recess.

In one embodiment, the central portion does not comprise a recess.

The plurality of recesses can be arranged in a substantially symmetricalor regular manner.

According to another embodiment, the plurality of recesses are arrangedin a substantially asymmetrical or irregular manner.

According to an embodiment, the plurality of recesses can be arranged ina substantially linear manner.

According to an embodiment, the plurality of recesses can be arranged ina substantially curved manner.

The plurality of sample wells can be arranged in an A×B format, whereinA and B are perpendicular axes, and the number of wells along the A axiscan be greater than, less than, or equal to the number of wells alongthe B axis.

According to an embodiment, the number of wells along the A axis or Baxis is at least 2.

The number of wells along the A axis or B axis can be between 2 and 15.

According to an embodiment, at least one of the plurality of samplewells is connected to another sample well of the plurality of sampleswells by a frangible region.

The sample plate can comprise a base comprising a docking portion forsecuring the sample plate to a corresponding docking portion of a plateframe holder.

According to an embodiment, the sample plate can further comprise abead.

The bead is can be attached to a probe.

The probe is can be a nucleic acid, antibody, antibody fragment,protein, peptide, aptamer, or chemical compound. According to anembodiment, the probe is an oligonucleotide.

In some embodiments, the sample plate comprises a plurality of probes,wherein a subset of the plurality of probes differs from another subsetof the plurality of probes.

In some embodiments, the plurality of probes comprise at least 3different probes.

Also provided herein are bead dispensing systems comprising:

a bead dispenser;

a sample plate comprising a sample well, wherein the sample wellcomprises a base portion, wherein the base portion comprises a recessand the recess comprises a diameter less than a diameter of a beaddispensed into the sample well; and

a control system configured to control dispensing of the bead from thebead dispenser into the sample plate.

The bead dispenser can comprise:

a syringe body comprising an annular chamber surrounding a longitudinalbore, wherein the annular chamber is configured to channel a reagentbead within the annular chamber towards a chamber provided in the bore;

a plunger provided within the longitudinal bore; and

a barrel or nozzle;

wherein the plunger is configured to dispense a bead from the chamberinto the barrel or nozzle.

The bead dispenser can be configured to dispense a plurality of beadsautomatically.

Also disclosed herein are methods of dispensing beads comprising:

providing a bead dispenser comprising a bead;

providing a sample plate comprising a sample well, wherein the samplewell comprises a base portion, wherein the base portion comprises arecess, wherein the recess comprises a diameter less than a diameter ofthe bead; and

controlling the dispensing of the bead from the bead dispenser into thesample plate.

The dispensing can be performed automatically.

Disclosed herein are kits for detecting an analyte comprising:

a plurality of beads; and

sample plate comprising a sample well, wherein the sample well comprisesa base portion, wherein the base portion comprises a recess, wherein therecess comprises a diameter less than a diameter of a bead of theplurality of beads.

The plurality of beads can comprise one or more probes.

The probe can comprise a nucleic acid, antibody, antibody fragment,protein, peptide, aptamer, or chemical compound.

According to an embodiment, the probe is an oligonucleotide.

Also disclosed herein are methods of detecting an analyte comprising:

adding a sample to a sample plate comprising a sample well, wherein thesample well comprises a base portion, wherein the base portion comprisesa recess, wherein the recess comprises a probe and the recess comprisesa diameter less than a diameter of a bead comprising the probe; anddetecting binding of an analyte in the sample with the probe.

The sample plate can comprise a plurality of probes and a plurality ofanalytes can be detected.

A plurality of samples can be added to the sample plate.

A sample plate is disclosed comprising one or more sample wells, whereinthe one or more sample wells comprise a base portion and one or morepockets or recesses provided in the base portion, wherein the one ormore pockets or recesses comprise a bore having a tapered sectionwherein, in use, a reagent bead or microsphere is substantially retainedor secured within the bore by the tapered section.

In some embodiments, the bore having the tapered section is not ashallow or small depression in which a reagent bead or microsphere restsbut in which the reagent bead or microsphere is not substantiallyretained or secured.

In some embodiments, in use, a reagent bead or microsphere issubstantially retained or secured within the bore by an interference orfriction fit with the tapered section of the bore.

In some embodiments, reagent beads are inserted into a sample platehaving a plurality of tapered holes or sections which act to firmlysecure or lock the reagent beads in position once inserted. A presetforce can be used to insert the reagent beads. The preset force can besufficient to compress the reagent bead and/or to deform the taperedsection of the bore so as to create or enhance the interference orfriction fit with the tapered section of the bore.

The sample plates disclosed herein can be particularly robust duringmanufacture and in subsequent processing stages including the stage ofinserting reagent beads into the tapered holes and subsequent handlingand processing of the sample plate. In some embodiments, the reagentbeads inserted into a sample plate are not free to move in any directionand essentially become a fixed part of the sample plate. In someembodiments, the angle of the taper can be arranged so that reagentbeads are locked or are otherwise firmly secured into the holes.

In some embodiments, in use, a reagent bead can be substantiallyretained or secured within the bore if the sample plate (i.e. the planeof the sample plate) is tipped by, for example more than about 10°, 20°,30°, 40°, 50°, 60°, 70°, 80°, or 90° to horizontal, or is inverted.

In some embodiments, the opening to the bore and/or cross-sectionalshape of the bore (i.e. at a location intermediate the opening to thebore and the base of the bore) is circular. In some embodiments, theopening and/or cross-sectional shape of the bore can be substantiallycircular, elliptical, oblong, triangular, square, rectangular,pentagonal, hexagonal, septagonal, octagonal, nonagonal, decagonal orpolygonal.

In some embodiments, the diameter of the opening of the bore is selectedfrom the group consisting of: (i) <0.5 mm; (ii) 0.5-1.0 mm; (iii)1.0-1.5 mm; (iv) 1.5-2.0 mm; (v) 2.0-2.5 mm; (vi) 2.5-3.0 mm; (vii)3.0-3.5 mm; (viii) 3.5-4.0 mm; (ix) 4.0-4.5 mm; (x) 4.5-5.0 mm; (xi)<5.0 mm; and (xii) >5.0 mm.

In some embodiments, the diameter of the opening of the bore is greaterthan the diameter of the reagent bead or microsphere. In someembodiments, the opening of the bore has a cross-sectional shape that isother than circular. In some embodiments, the smallest span of thecross-sectional shape of the bore at the opening is greater than thediameter of the reagent bead or microsphere.

In one embodiment, a diameter of the bore at a location intermediate theopening of the bore and the base of the bore can be at least 5% smallerthan the diameter of the reagent bead or microsphere and/or is at least5% smaller than the diameter of the opening of the bore. In someembodiments, the bore has a cross-sectional shape that is other thancircular and the smallest span of the cross-sectional shape of the boreat a location intermediate the opening of the bore and the base of thebore is at least 5% smaller than the diameter of the reagent bead ormicrosphere and/or is at least 5% smaller than the diameter of theopening of the bore.

In some embodiments, a diameter of the bore at a location intermediatethe opening of the bore and the base of the bore is selected from thegroup consisting of: (i) <0.5 mm; (ii) 0.5-1.0 mm; (iii) 1.0-1.5 mm;(iv) 1.5-2.0 mm; (v) 2.0-2.5 mm; (vi) 2.5-3.0 mm; (vii) 3.0-3.5 mm;(viii) 3.5-4.0 min; (ix) 4.0-4.5 mm; (x) 4.5-5.0 mm; (xi) <5.0 mm; and(xii) >5.0 mm.

In some embodiments, the tapered section of the bore is substantiallylinearly tapered. For example, the diameter or circumference of the borevaries (e.g. decreases) substantially linearly with the depth of thebore. In some embodiments, the bore has a cross-sectional shape that isother than circular and a cross-sectional dimension (e.g. the smallestspan of the cross-sectional shape of the bore) or the perimeter of thecross-sectional shape of the bore varies (e.g. decreases) substantiallylinearly with the depth of the bore.

In some embodiments, the reagent beads are opaque and signal is onlydetected from the top of the bead. In some embodiments, the bottom ofthe bead below a press fit or interference fit line does not come intocontact with sample fluid. In some embodiments, in use, a reagent beadforms a substantially fluid-tight seal with either the cylindrical ortapered section of the bore so as to substantially prevent fluid fromflowing from the sample well past the reagent bead. A sample plate withinserted reagent beads, according to some embodiments, thereforeresembles an empty conventional sample well.

In some embodiments, the reagent beads protrude above the bottom of thesample well. In some embodiments, the reagent beads do not form a moatregion around the upper portion of the bead. In some embodiments, thereagent beads do not trap fluid.

In some embodiments, the reagent beads can be arranged so as not toprotrude above the bottom of the sample well. In some embodiments,reagent beads are protected and are not susceptible to damage throughhandling, pipetting or washing. In some embodiments, the bore depth atwhich the diameter of the bore becomes less than the diameter of thereagent bead is equal to or greater than the radius of the reagent bead.In some embodiments, the reagent beads do not protrude above the bottomof the sample well. In some embodiments, the bore has a cross-sectionalshape that is other than circular and the bore depth at which thesmallest span of the cross-sectional shape of the bore becomes less thanthe diameter of the reagent bead is equal to or greater than the radiusof the reagent bead.

Beads can be pressed or inserted into the pockets, recesses or boresformed in the base portion of the sample wells. The tops of the reagentbeads once inserted can protrude above the bottom of the sample well, oralternatively, can be flush or level with the bottom of the sample well.

In some embodiments, a 2 mm bead can be arranged to protrude 0.5 mmabove the bottom of the base portion of the sample well. According tosome embodiments, one or more of the reagent beads can be arranged toprotrude a distance of 0-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%,30-35%, 35-40% or >40% of the diameter of the bead above the bottom ofthe base portion of the sample well.

According to some embodiments, the depth of the bore is selected fromthe group consisting of: (i) <0.5 mm; (ii) 0.5-1.0 mm; (iii) 1.0-1.5 mm;(iv) 1.5-2.0 mm; (v) 2.0-2.5 mm; (vi) 2.5-3.0 mm; (vii) 3.0-3.5 mm;(viii) 3.5-4.0 mm; (ix) 4.0-4.5 mm; (x) 4.5-5.0 mm; (xi) <5.0 mm; and(xii) >5.0 mm.

In some embodiments, in use, the reagent bead does not contact the baseof the bore. In some embodiments, a reagent bead may contact the base ofthe bore.

In some embodiments, the reagent beads can be inserted so that they areflush with the bottom of the well and the sample plate can be used withknown automated microplate processing systems requiring only minimalhardware modifications. Furthermore, the sample well according to suchan embodiment is essentially a cylinder having proportions which aresimilar to that of a well of a conventional microplate so the fluid andother handling characteristics of the sample well are well known.Processing steps according to such an embodiment such as pipetting,mixing, washing and incubation follow the same type of fluidcharacteristics that conventional microplates go through.

The sample plate according can have a fluid capacity of approximately800 μl. In some embodiments, in use, only a small fraction of the totalfluid capacity of a sample well is required in order to cover all thereagent beads disposed in the base of the sample plate.

In some embodiments, fluid can be dispensed directly into the centre orcentral region of a sample well and the sample plate may be arranged sothat no pockets, recesses or bores for securing reagent beads arearranged in the central region of the sample well. In some embodiments,reagent which coats the reagent beads is not inadvertently washed offthe reagent beads by the force of the fluid jet from a wash head orpipette tip.

The sample plate according some embodiments, enables multiple tests tobe carried out in a single sample well. This can be achieved byinserting different reagent beads into separate bores in the same samplewell thereby enabling multiplexing to be performed. In some embodiments,reagent beads can be pressed into tapered or non-tapered holes in thebase of the well as desired which results in a high degree offlexibility and the ability to use the entire sample well with a highefficiency.

In some embodiments, a sample plate can comprise one or more 12 mmdiameter sample wells. Each sample well can have a cross sectionalsurface area of 58 mm2 and in total 54 sample wells of this size can befitted into a conventional microplate footprint. Within each sample wella varied number of beads can be inserted. The bores in a sample well canhave different diameters to accommodate different size reagent beads ifdesired.

According to other embodiments, one or more sample wells may comprise6×3.0 mm diameter pockets, recesses or bores, 10×2.0 mm diameterpockets, recesses or bores or 21×1.75 mm pockets, recesses or bores. Thecentral region of the sample well can be kept free of pockets, recessesor bores. The pockets, recesses or bores may be arranged in a circle ortwo or more concentric circles or other patterns about the centralregion of the sample well.

According to an embodiment, a sample plate having an array of 9×6 samplewells may be provided. If six pockets, recesses or bores are providedper sample well, then the sample plate can accommodate 324 reagent beadsper plate. If 10 pockets, recesses or bores are provided per samplewell, then the sample plate can accommodate 540 reagent beads per plate.If 21 pockets, recesses or bores are provided per sample well, then thesample plate can accommodate 1134 reagent beads per plate.

In some embodiments, the sample plate according to the present inventionis relatively simple to manufacture compared with other knownarrangements. The sample plate can be manufactured by moulding using anopen and shut tool so that the manufacturability is high and reliable.The injection mould tool design used to form the sample plates is simpleand does not require the use of undercuts or thin features to mould. Asa result, the production of sample plates having different formats canbe readily achieved. A tool that produces a sample well with six pocketsor bores can be readily adapted to produce a sample well having adifferent number (e.g. 21) of pockets.

In some embodiments, validation of different well designs and formatscan be achieved simply since the test protocols can remain essentiallythe same. In some embodiments, pipetting and incubation do not changeand the washing procedure would only requires, at most, a minoralteration to the aspirate routine.

The tapered section or bore can have a taper selected from the groupconsisting of: (i) <0.5°; (ii) 0.5°; (iii) 0.5-1°; (iv) 1-2°; (v) 2-4°;(vi) 4-6°; (vii) 6-8°; (viii) 8-10°; and (ix) >10°. Alternatively,through holes or bores provided in the base portion may be cylindricaland non-tapered.

In some embodiments, arrangement of the pockets or recesses provided inthe base portion may comprise a chamber having a retention member,membrane, lip or annular portion. A reagent bead or microsphere may beinserted, in use, past or through the retention member, membrane, lip orannular portion into the chamber and may be substantially retained orsecured within the chamber by the retention member, membrane, lip orannular portion.

The one or more pockets, recesses or bores can comprise a countersunk orenlarged portion for facilitating the insertion of a reagent bead ormicrosphere into one or more of the pockets, recesses or bores.

The one or more sample wells can comprise at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 pockets orrecesses each comprising a bore having a tapered or non-tapered sectionand which are each arranged and adapted to receive, in use, a reagentbead or microsphere.

The one or more pockets, recesses or bores provided in the base portioncan be arranged: (i) circumferentially around a central portion of thesample well; and/or (ii) with a plurality of pockets or recessesarranged circumferentially around one more central pockets or recesses;and/or (iii) in a substantially close-packed manner; and/or (iv) in asubstantially symmetrical or asymmetrical manner; and/or (v) in asubstantially linear or curved manner; and/or (vi) in a substantiallyregular or irregular manner; and/or (vii) in an array; and/or (viii) ina circle or two or more concentric circles with no pocket, recess orbore located at the centre of the base portion.

The sample plate can be fabricated or otherwise made from polystyrene.

The sample plate may comprise either a strip or an array format. Forexample, according to an embodiment the sample plate may comprise a 6×1strip of sample wells. According to another embodiment the sample platemay comprise nine 6×1 sample strips of sample wells.

According to an embodiment one or more of the sample wells may beinterconnected to one or more other sample wells by one or morefrangible regions or connections so that the sample plate can beseparated by a user into a plurality of smaller sample plates, samplestrips or individual sample wells. This enables a sample plate to besnapped or broken into a plurality of smaller sample plates. Forexample, a 6×1 strip of sample wells may be snapped into six individualsample wells or into two 3×1 sample strips.

According to an embodiment individual sample wells, sample strips andsample plates are made from polypropylene. Sample wells, sample stripsand sample plates can be made from a non-binding material such aspolypropylene to ensure non-specific binding in the well is kept to aminimum.

A plate frame arranged to hold a plurality of sample wells, samplestrips or one or more sample plates can be made from a plastic such asAcrylonitrile Butadiene Styrene (“ABS”). The plate frame can be madefrom a material which provides high rigidity and which ensures thatsample wells, sample strips or one or more sample plates are heldsecurely in place and remain flat after sample wells, sample strips orsample plates are secured into the plate frame. The plate frame can besufficiently robust to withstand handling by a user.

One or more of the sample wells may be interconnected to one or moreother sample wells by one or more frangible regions or connections sothat the sample plate can be separated by a user into a plurality ofsmaller sample plates, sample strips or individual sample wells.

According to an aspect of the disclosure, there is provided a computerprogram executable by the control system of an automated apparatus, theautomated apparatus comprising one or more reagent bead or microspheredispensers, the computer program being arranged to cause the controlsystem:

(i) to control the dispensing of reagent beads or microspheres from theone or more reagent bead or microsphere dispensers into one or moresample wells of a sample plate as disclosed above.

According to an aspect of the disclosure there is provided a computerreadable medium comprising computer executable instructions stored onthe computer readable medium, the instructions being arranged to beexecutable by a control system of an automated apparatus, the automatedapparatus comprising one or more reagent bead or microsphere dispensers,the computer program being arranged to cause the control system:

(i) to control the dispensing of reagent beads or microspheres from theone or more reagent bead or microsphere dispensers into one or moresample wells of a sample plate as disclosed above.

The computer readable medium can be selected from the group consistingof: (i) a ROM; (ii) an EAROM; (iii) an EPROM; (iv) an EEPROM; (v) aflash memory; (vi) an optical disk; (vii) a RAM; and (viii) a hard diskdrive.

In some embodiments, at least some or substantially all of the reagentbeads or microspheres which are dispensed, in use, into one or more ofthe pockets, recesses or bores carry or comprise a reagent, wherein thereagent is arranged and adapted: (i) to analyze samples; and/or (ii) toanalyze samples by nucleic acid amplification reactions; and/or (iii) toanalyze samples by polymerase chain reactions (PCR); and/or (iv) toanalyze samples by an immunoassay process; and/or (v) to analyze samplesby using a hybridization probe technique.

In some embodiments, at least some or substantially all of the reagentbeads or microspheres which are dispensed, in use, into one or more ofthe pockets, recesses or bores comprise polystyrene, plastic or apolymer.

The sample plate disclosed herein can comprise one or more beads. Thebead can be a microparticle, particle, microsphere, or grammaticalequivalents. The bead composition is dependent on the type of assaybeing performed. The bead may be composed of plastics, ceramics, glass,polystyrene, methylstyrene, acrylic polymers, paramagnetic materials,thoria sol, carbon graphite, titanium dioxide, latex or cross-linkeddextrans such as Sepharose, cellulose, nylon, cross-linked micelles,Teflon or any combination thereof. In one embodiment, a bead comprisespolystyrene, plastic, a polymer, or a combination thereof. In anotherembodiment, a bead comprises a ferrous or magnetic coating or has aferrous or magnetic property. In yet another embodiment, a beadcomprises an anti-static coating or has an anti-static property. Thebead used in the sample plate reagent beads can be translucent, slightlytranslucent, or opaque. Commercially available beads can also be used.

The beads need not be spherical and may be of irregular shape. Inaddition, the beads may be porous. The bead size may range fromnanometers to millimeters. The bead may have a diameter of at least 0.1mm. The bead may have a diameter of between 0.1 mm and 10 mm. In oneembodiment, the bead may have a diameter of greater than about 0.5 mm;0.5-1.0 mm; 1.0-1.5 mm; 1.5-2.0 mm; 2.0-2.5 mm; 2.5-3.0 mm; 3.0-3.5 mm;3.5-4.0 mm; 4.0-4.5 mm; 4.5-5.0 mm; or greater than about 5.0 mm. Thebead may have a diameter greater than, equal to, or less than thediameter of a recess, pocket, or bore of a sample well. For example, thebead may have a diameter less than the diameter of a recess, pocket, orbore of a sample well, wherein the recess, pocket or bore comprises atapered section. In yet another embodiment, the bead may have a diametergreater than the diameter of a recess, pocket, or bore of a sample well.For example, the recess, pocket, or bore may not comprise a taperedsection. The diameter of a bead to be deposited, or present, in thesample plate, can be at least about 5, 10, 15, 20, 35, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% greater than thediameter of a recess of the sample plate. In one embodiment, the beadpresent in a sample plate does not touch the bottom of a sample plate,such as a base portion of a sample well.

A bead within the sample plate may comprise a reagent or probe, or becoated with a reagent or probe. The reagent or probe can be used toanalyze a sample, such as by detecting an analyte. The probe or reagentcan be attached to the bead. The attachment can be a covalent ornon-covalent interaction. The probe can be a nucleic acid, antibody,antibody fragment, protein, peptide, aptamer, or chemical compound. Forexample, the probe can be an oligonucleotide. In one embodiment, theprobe can be used to detect an analyte in a biological sample. In yetanother embodiment, the probe can be used to for drug screening. Forexample, a library of compounds or antibodies can be screened for itsbinding ability to a protein or nucleic acid probe.

The probe can be used to provide detect a biomarker for a diagnosis orprognosis of a disease or condition, drug response or potential drugresponse, or for monitoring the progression of a disease or condition.For example, the probe can be an antibody or fragment thereof that isused to detect an antigen that is a biomarker for cancer. In anotherembodiment, the probe can be an antigen, peptide or protein, which isused to detect an antibody in a sample, which can be an indicative of adisease or condition.

The sample plate disclosed herein can comprise a plurality of probes,wherein a subset of the plurality differs from another subset of theplurality. The plurality of probes can be attached to beads. Thedifferent probes can be used to detect different analytes, thus allowingmultiplexing with the sample plates disclosed herein. The sample platecan comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 different probes. The probes can be of the sametype (for example, different antibodies) or of a different type (forexample, a combination of nucleic acid probe(s) and antigen(s)).

According to an embodiment the one or more of the reagent bead ormicrosphere dispensers can comprise a tube containing, in use, aplurality of reagent beads or microspheres.

The apparatus can further comprise one or more sensors for sensingwhether or not one or more reagents beads have been dispensed from oneor more of the reagent bead or microsphere dispensers.

The apparatus can further comprise a translation stage for moving thesample plate relative to one or more reagent bead or microspheredispensers.

The control system can be arranged and adapted to control thetranslation stage so that one or more reagent beads or microspheres froma reagent bead or microsphere dispenser are dispensed sequentially intodifferent reagent bead or microsphere receiving chambers by moving thesample plate relative to the reagent bead or microsphere dispenser.

According to an embodiment the apparatus further comprises a fluiddispensing device for dispensing fluid into the sample wells of a sampleplate.

The fluid dispensing device can be arranged and adapted to dispense x mlof fluid at a time into the one or more fluid receiving areas of one ormore sample wells, wherein x is preferably selected from the groupconsisting of: (i) <10; (ii) 10-20; (iii) 20-30; (iv) 30-40; (v) 40-50;(vi) 50-60; (vii) 60-70; (viii) 70-80; (ix) 80-90; (x) 90-100; (xi)100-110; (xii) 110-120; (xiii) 120-130; (xiv) 130-140; (xv) 140-150;(xvi) 150-160; (xvii) 160-170; (xviii) 170-180; (xix) 180-190; (xx)190-200; and (xxi) >200.

In some embodiments, the fluid dispensing device is arranged and adaptedto dispense about 1 μL, μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, 15 μL, 16 μL, 17 μL, 18 μL, 19μL, 20 μL, 21 μL, 22 μL, 23 μL, 24 μL, 25 μL, 26 μL, 27 μL, 28 μL, 29μL, 30 μL, 31 μL, 32 μL, 33 μL, 34 μL, 35 μL, 36 μL, 37 μL, 38 μL, 39μL, 40 μL, 41 μL, 42 μL, 43 μL, 44 μL, 45 μL, 46 μL, 47 μL, 48 μL, 49μL, 50 μL, 51 μL, 52 μL, 53 μL, 54 μL, 55 μL, 56 μL, 57 μL, 58 μL, 59μL, 60 μL, 61 μL, 62 μL, 63 μL, 64 μL, 65 μL, 66 μL, 67 μL, 68 μL, 69μL, 70 μL, 71 μL, 72 μL, 73 μL, 74 μL, 75 μL, 76 μL, 77 μL, 78 μL, 79μL, 80 μL, 81 μL, 82 μL, 83 μL, 84 μL, 85 μL, 86 μL, 87 μL, 88 μL, 89μL, 90 μL, 91 μL, 92 μL, 93 μL, 94 μL, 95 μL, 96 μL, 97 μL, 98 μL, 99μL, 100 μL, 101 μL, 102 μL, 103 μL, 104 μL, 105 μL, 106 μL, 107 μL, 108μL, 109 μL, 110 μL, 111 μL, 112 μL, 113 μL, 114 μL, 115 μL, 116 μL, 117μL, 118 μL, 119 μL, 120 μL, 121 μL, 122 μL, 123 μL, 124 μL, 125 μL, 126μL, 127 μL, 128 μL, 129 μL, 130 μL, 131 μL, 132 μL, 133 μL, 134 μL, 135μL, 136 μL, 137 μL, 138 μL, 139 μL, 140 μL, 141 μL, 142 μL, 143 μL, 144μL, 145 μL, 146 μL, 147 μL, 148 μL, 149 μL, 150 μL, 151 μL, 152 μL, 153μL, 154 μL, 155 μL, 156 μL, 157 μL, 158 μL, 159 μL, 160 μL, 161 μL, 162μL, 163 μL, 164 μL, 165 μL, 166 μL, 167 μL, 168 μL, 169 μL, 170 μL, 171μL, 172 μL, 173 μL, 174 μL, 175 μL, 176 μL, 177 μL, 178 μL, 179 μL, 180μL, 181 μL, 182 μL, 183 μL, 184 μL, 185 μL, 186 μL, 187 μL, 188 μL, 189μL, 190 μL, 191 μL, 192 μL, 193 μL, 194 μL, 195 μL, 196 μL, 197 μL, 198μL, 199 μL, 200 μL, or more fluid at a time into the one or more fluidreceiving areas of one or more sample wells.

According to an embodiment the apparatus further comprises an imageanalysis device or camera for determining whether or not a reagent beador microsphere has been dispensed or is otherwise present in a pocket,recess or bore of the sample plate.

The sample plate can have a first color (or is transparent) and thereagent beads or microspheres can have a second different color whichcontrasts with the first color (or transparency) in order to facilitatevisual detection of the presence or absence of a reagent bead ormicrosphere in a pocket, recess or bore of the sample plate.

According to an embodiment the sample plate may further comprise aluminescence or fluorescence marker.

The apparatus may further comprise a luminescence or fluorescencedetecting device for determining whether or not a reagent bead ormicrosphere has been dispensed or is otherwise present in a pocket,recess or bore of the sample plate by determining whether or not areagent bead or microsphere obstructs or partially obstructs theluminescence or fluorescence marker.

The apparatus may further comprise a magnetic and/or electrical and/orcapacitive and/or mechanical sensor for sensing whether or not a reagentbead or microsphere has been dispensed or is otherwise present in apocket, recess or bore of a sample plate.

The control system may determine the number of reagent beads ormicrospheres present and/or the number of reagent beads or microspheresabsent and/or the number of reagent beads or microspheres dispensedand/or the number of reagent beads or microspheres desired to bedispensed in a sample well.

According to an embodiment the control system may measure and/or adjustthe volume of fluid dispensed or desired to be dispensed into a samplewell dependent upon the number of reagent beads or microspheresdetermined to be present and/or absent and/or dispensed and/or desiredto be dispensed in the sample well.

The control system may be arranged and adapted to ensure that the uppersurface of at least some or substantially all reagent beads ormicrospheres located in the bores of a sample well are at leastpartially or fully immersed by a fluid when the fluid is dispensed intothe sample well.

The control system is preferably arranged and adapted to ensure that theheight of fluid dispensed into a sample well remains substantiallyconstant irrespective of the number of reagent beads or microspherespresent, absent, dispensed or desired to be dispensed into the samplewell.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments of the present invention will now be described, byway of example only, and with reference to the accompanying drawings inwhich:

FIG. 1 shows a sample well of a sample plate according to an embodimentof the present invention;

FIG. 2A shows a plan view of a sample well of a sample plate accordingto an embodiment, FIG. 2B shows in greater detail the bottom of a samplewell according to an embodiment and FIG. 2C shows a reagent bead ormicrosphere dispensed in a pocket of a sample well according to anembodiment;

FIG. 3A shows a reagent bead or microsphere dispenser and FIG. 3B showsa cutaway view of the reagent bead or microsphere dispenser;

FIG. 4 shows an exploded view of the reagent bead or microspheredispenser;

FIG. 5 shows a microarrayer comprising a reagent bead or microspheresyringe pick-up device mounted on an x-y-z translation stage and engagedwith a reagent bead or microsphere dispenser above a sample plate;

FIG. 6 shows in greater detail a cutaway view of a reagent bead ormicrosphere syringe pick-up device attached to a reagent bead ormicrosphere dispenser;

FIG. 7A shows a reagent bead or microsphere dispenser being transportedby a reagent bead or microsphere syringe pick-up device and FIG. 7Bshows a reagent bead or microsphere in the process of being dispensedfrom a reagent bead or microsphere dispenser by a plunger mechanismwhich is actuated by the reagent bead or microsphere syringe pick-updevice;

FIG. 8A shows a reagent bead or microsphere syringe in the process ofbeing ejected from the reagent bead or microsphere syringe pick-updevice and FIG. 8B shows the reagent bead or microsphere syringe havingbeen ejected from the reagent bead or microsphere pick-up device;

FIG. 9A shows nine sample strips loaded into a plate frame, wherein eachsample strip comprises a 6×1 array of sample wells and FIG. 9B shows aplate frame into which a sample plate or one or more sample strips maybe loaded;

FIG. 10A shows in greater detail a sample strip comprising six samplewells and FIG. 10B shows a sample strip comprising six sample wellsbeing loaded into a plate frame;

FIG. 11A shows a single well being loaded into a plate frame, FIG. 11Bshows in greater detail two sample wells connected by a break apartfeature, FIG. 11C shows a sample well having an end feature and FIG. 11Dshows a sample well having an ID and orientation tab;

FIG. 12A shows the underneath of a strip of sample wells, FIG. 12B showsa female alignment and retaining feature which helps to align a samplestrip or sample well with a plate frame and FIG. 12C shows acorresponding male alignment and retaining feature which is provided inthe base of the plate frame;

FIG. 13 shows a cross-sectional view of a strip of sample wells andshows an embodiment wherein the sample wells have a plurality of taperedbores wherein the angle of the taper is 6.0°;

FIG. 14A shows a further embodiment of the present invention whereinconical through holes are provided in the base portion of a sample plateand reagent beads are loaded from the rear of the sample plate and FIG.14B shows a sample plate according to a preferred embodiment wherein thesample plate has a cylindrical non-tapered through hole such thatreagent beads may be loaded or inserted from the top through the samplewell and are secured within the through hole by an interference fit;

FIG. 15 shows a sample strip comprising six sample wells wherein reagentbeads are fitted from the underneath of the sample plate;

FIG. 16 shows a cross sectional 3D view of the further embodimentshowing reagent beads located within a concave end portion of a throughhole;

FIG. 17 shows an embodiment wherein the base portion of a sample well issegmented into five segments and each base portion segment is arrangedat a different relative height so that there is no direct line of sightbetween reagent beads inserted into open through holes or recessesprovided in each base portion segment;

FIG. 18A shows a plan view of an embodiment wherein a relativelylow-height baffle divides a base portion into two sections so that thereis no direct line of sight between reagent beads inserted into openthrough holes or recesses provided in one section and reagent beadsinserted into open through holes or recesses provided in the othersection and FIG. 18B shows a 3D view of an embodiment wherein alow-height baffle separates the base portion into two sections so thatthere is no direct line of sight between reagent beads inserted intoopen through holes or recesses provided in one section and reagent beadsinserted into open through holes or recesses provided in the othersection; and

FIG. 19A shows a plan view of an embodiment wherein a relativelylow-height baffle divides a base portion into two sections so that thereis no direct line of sight between reagent beads inserted into openthrough holes or recesses provided in one section and reagent beadsinserted into open through holes or recesses provided in the othersection and FIG. 19B shows a 3D view of an embodiment wherein arelatively low-height baffle divides a base portion into two sections sothat there is no direct line of sight between reagent beads insertedinto open through holes or recesses provided in one section and reagentbeads inserted into open through holes or recesses provided in the othersection.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

DETAILED DESCRIPTION

An embodiment of the present invention will now be described withreference to FIG. 1. A sample plate is provided that can comprise aplurality of sample wells 19 (in one embodiment, a sample plate can beprovided which comprises only a single sample well 19). According to oneembodiment, the sample plate may comprise a 9×6 array of sample wells19. A single sample well 19 is shown in FIG. 1 for ease of illustration.Embodiments are also contemplated wherein the sample plate may comprisea strip of sample wells 19 e.g. the sample plate may comprise, forexample, a sample strip comprising an 1×9 or an 1×6 array of samplewells 19.

Each sample well 19 can comprise a plurality of pockets, recesses orbores 21 which are provided in the base of the sample well 19. In theparticular embodiment shown in FIG. 1 the sample well 19 comprises tenpockets, recesses or bores 21 which are formed or otherwise provided inthe base of a sample well 19. Other embodiments are contemplated whereina different number of pockets, recesses or bores 21 may be provided inthe base of the sample well 19. For example, according to alternativeembodiments at least some or all of the sample wells 19 provided in asample plate may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21 or >21 pockets, recesses or bores 21.

The pockets, recesses or bores 21 can be provided around the edge orperimeter of the sample well 19 and the centre or central region of thebase of the sample well 19 can be substantially flat and free frompockets, recesses or bores 21.

According to an embodiment a plurality of reagent beads or microsphereseach having a diameter of 1.75 or 2 mm may be loaded into a reagent beador microsphere dispenser. According to another embodiment a reagent beador microsphere dispensers may be provided which is arranged to handlereagent beads or microspheres having a diameter other than 1.75 mm or 2mm. Other embodiments are also contemplated wherein reagent beads ormicrospheres in a first reagent bead or microsphere dispenser may have afirst diameter and wherein reagent beads or microspheres in a seconddifferent reagent bead or microsphere dispenser may have a seconddifferent diameter. Other embodiments are also contemplated wherein thereagent beads or microspheres loaded into a particular reagent bead ormicrosphere dispenser may have a plurality or mixture of differentdiameters.

The reagent beads or microspheres may be pre-loaded or pre-inserted intothe pockets, recesses or bores 21 by a sample plate manufacturer.Alternatively, an end-user may load or insert the reagent beads ormicrospheres into the pockets, recesses or bores 21.

The reagent beads or microspheres can comprise a polystyrene, plastic orpolymer core. The reagent beads or microspheres may be coated with areagent (e.g. an antibody or antigen) which can be used to analyzesamples. According to an embodiment the reagent may be used to analyzesamples by polymerase chain reactions (PCR) or as part of an immunoassayprocedure. Alternatively, according to another embodiment the reagentmay comprise a DNA or RNA sequence which is used as a hybridizationprobe to detect the presence of complementary DNA or RNA sequences in asample. The reagent beads or microspheres may also be coated with ananti-static coating or may have an anti-static property.

A fluid to be tested can be dispensed into a sample well 19 of a sampleplate. The fluid may, for example, comprise a sample of blood, serum,saliva or urine taken from a patient.

According to an embodiment, about 10-200 ml of fluid sample may bedispensed into each sample well 19 of a sample plate, e.g., about 10 mL,11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 21mL, 22 mL, 23 mL, 24 mL, 25 mL, 26 mL, 27 mL, 28 mL, 29 mL, 30 mL, 31mL, 32 mL, 33 mL, 34 mL, 35 mL, 36 mL, 37 mL, 38 mL, 39 mL, 40 mL, 41mL, 42 mL, 43 mL, 44 mL, 45 mL, 46 mL, 47 mL, 48 mL, 49 mL, 50 mL, 51mL, 52 mL, 53 mL, 54 mL, 55 mL, 56 mL, 57 mL, 58 mL, 59 mL, 60 mL, 61mL, 62 mL, 63 mL, 64 mL, 65 mL, 66 mL, 67 mL, 68 mL, 69 mL, 70 mL, 71mL, 72 mL, 73 mL, 74 mL, 75 mL, 76 mL, 77 mL, 78 mL, 79 mL, 80 mL, 81mL, 82 mL, 83 mL, 84 mL, 85 mL, 86 mL, 87 mL, 88 mL, 89 mL, 90 mL, 91mL, 92 mL, 93 mL, 94 mL, 95 mL, 96 mL, 97 mL, 98 mL, 99 mL, 100 mL, 101mL, 102 mL, 103 mL, 104 mL, 105 mL, 106 mL, 107 mL, 108 mL, 109 mL, 110mL, 111 mL, 112 mL, 113 mL, 114 mL, 115 mL, 116 mL, 117 mL, 118 mL, 119mL, 120 mL, 121 mL, 122 mL, 123 mL, 124 mL, 125 mL, 126 mL, 127 mL, 128mL, 129 mL, 130 mL, 131 mL, 132 mL, 133 mL, 134 mL, 135 mL, 136 mL, 137mL, 138 mL, 139 mL, 140 mL, 141 mL, 142 mL, 143 mL, 144 mL, 145 mL, 146mL, 147 mL, 148 mL, 149 mL, 150 mL, 151 mL, 152 mL, 153 mL, 154 mL, 155mL, 156 mL, 157 mL, 158 mL, 159 mL, 160 mL, 161 mL, 162 mL, 163 mL, 164mL, 165 mL, 166 mL, 167 mL, 168 mL, 169 mL, 170 mL, 171 mL, 172 mL, 173mL, 174 mL, 175 mL, 176 mL, 177 mL, 178 mL, 179 mL, 180 mL, 181 mL, 182mL, 183 mL, 184 mL, 185 mL, 186 mL, 187 mL, 188 mL, 189 mL, 190 mL, 191mL, 192 mL, 193 mL, 194 mL, 195 mL, 196 mL, 197 mL, 198 mL, 199 mL, 200mL. According to the preferred embodiment less fluid may be dispensedinto each sample well 19 compared with a conventional sample plate.

According to another embodiment, about 10-200 μL of fluid sample may bedispensed into each sample well 19 of a sample plate, e.g., about 10 μL,11 μL, 12 μL, 13 μL, 14 μL, 15 μL, 16 μL, 17 μL, 18 μL, 19 μL, 20 μL, 21μL, 22 μL, 23 μL, 24 μL, 25 μL, 26 μL, 27 μL, 28 μL, 29 μL, 30 μL, 31μL, 32 μL, 33 μL, 34 μL, 35 μL, 36 μL, 37 μL, 38 μL, 39 μL, 40 μL, 41μL, 42 μL, 43 μL, 44 μL, 45 μL, 46 μL, 47 μL, 48 μL, 49 μL, 50 μL, 51μL, 52 μL, 53 μL, 54 μL, 55 μL, 56 μL, 57 μL, 58 μL, 59 μL, 60 μL, 61μL, 62 μL, 63 μL, 64 μL, 65 μL, 66 μL, 67 μL, 68 μL, 69 μL, 70 μL, 71μL, 72 μL, 73 μL, 74 μL, 75 μL, 76 μL, 77 μL, 78 μL, 79 μL, 80 μL, 81μL, 82 μL, 83 μL, 84 μL, 85 μL, 86 μL, 87 μL, 88 μL, 89 μL, 90 μL, 91μL, 92 μL, 93 μL, 94 μL, 95 μL, 96 μL, 97 μL, 98 μL, 99 μL, 100 μL, 101μL, 102 μL, 103 μL, 104 μL, 105 μL, 106 μL, 107 μL, 108 μL, 109 μL, 110μL, 111 μL, 112 μL, 113 μL, 114 μL, 115 μL, 116 μL, 117 μL, 118 μL, 119μL, 120 μL, 121 μL, 122 μL, 123 μL, 124 μL, 125 μL, 126 μL, 127 μL, 128μL, 129 μL, 130 μL, 131 μL, 132 μL, 133 μL, 134 μL, 135 μL, 136 μL, 137μL, 138 μL, 139 μL, 140 μL, 141 μL, 142 μL, 143 μL, 144 μL, 145 μL, 146μL, 147 μL, 148 μL, 149 μL, 150 μL, 151 μL, 152 μL, 153 μL, 154 μL, 155μL, 156 μL, 157 μL, 158 μL, 159 μL, 160 μL, 161 μL, 162 μL, 163 μL, 164μL, 165 μL, 166 μL, 167 μL, 168 μL, 169 μL, 170 μL, 171 μL, 172 μL, 173μL, 174 μL, 175 μL, 176 μL, 177 μL, 178 μL, 179 μL, 180 μL, 181 μL, 182μL, 183 μL, 184 μL, 185 μL, 186 μL, 187 μL, 188 μL, 189 μL, 190 μL, 191μL, 192 μL, 193 μL, 194 μL, 195 μL, 196 μL, 197 μL, 198 μL, 199 μL, or200 μL.

According to an embodiment a control system may be used to determine thelocation and/or type of reagent beads or microspheres which have beendispensed into the bores 21 of a sample well 19. Alternatively, thereagent beads or microspheres may be pre-loaded into the bores 21 of thesample wells 19. The control system may also determine into which bores21 (if any) additional reagent beads or microspheres need to bedispensed. Once sample fluid has been dispensed into a sample well 19,the control system may check that an appropriate amount of sample fluidhas been dispensed and that all the reagent beads or microspheres are atleast partially or are fully immersed by the sample fluid.

The volume of sample fluid to be dispensed into a sample well 19 maydepend upon the number of bores 21 formed within a sample well 19, thediameter of the reagent beads or microspheres which are dispensed orpre-loaded into the bores 21 and the extent to which reagent beads ormicrospheres protrude into the bottom of the sample well 19. The controlsystem may be used to vary the amount of sample fluid dispensed into asample well 19 so that reagent beads or microspheres are immersed insample fluid to a substantially constant depth irrespective of thenumber of bores present in a sample well 19, the diameter of the reagentbeads or microspheres or the extent to which the reagent beads ormicrospheres protrude into the base of the sample well 19.

Different formats of sample plates may be provided. For example, asample plate may comprise a two dimensional array of sample wells 19e.g. the sample plate may comprise a 4×4, 4×6, 4×8, 4×10, 4×12, 6×6,6×8, 6×10, 6×12, 8×8, 8×10, 8×12, 10×10, 10×12 or 12×12 array of samplewells 19. According to other embodiments the sample plate may comprise asingle dimensional strip of sample wells 19 e.g. the sample plate maycomprise a 4×1, 6×1, 8×1, 10×1 or 12×1 strip of sample wells 19. Yetfurther embodiments are contemplated wherein the sample wells 19 may beprovided in a format other than in an array or strip.

At least some or all of the pockets, recesses or bores 21 which areprovided in the base of a sample well 19 may comprise a bore which isoptionally tapered along at least a portion or substantially the wholeof its length. The pockets, recesses or bores 21 may, for example, bearranged to have a 6° taper. According to an embodiment the top (orreagent bead or microsphere receiving portion) of a tapered bore mayhave a diameter of 1.82 mm. The base of the sample well 19 surroundingthe bore may be arranged to have a countersunk portion in order tofacilitate the insertion of a reagent bead or microsphere 20A; 20B intothe pocket, recess or bore 21. According to an embodiment the outerdiameter of the countersunk portion may be 2.25 mm.

FIG. 2A shows a plan view of a sample well 19 and portions of twoadjacent sample wells 19 which are provided in a sample plate. Thesample wells shown in FIG. 2A form part of an array of sample wells 19which are provided in the sample plate. Each of the sample wells 19comprise ten pockets, recesses or bores 21 which are disposed in thebottom or base portion of the sample well 19. In use reagent beads ormicrospheres can be inserted into each of the pockets, recesses or bores21 of a sample well 19 and with the embodiment shown in FIGS. 2A-2C thereagent beads or microspheres are preferably secured in the pockets,recesses or bores 21 by virtue of the diameter of the bore tapering andbecoming restricted.

FIG. 2B shows in greater detail the bottom of a sample well 19 and showsa plurality of pockets, recesses or bores 21 provided in the bottomportion of the sample well 19 each of which are arranged and adapted toreceive a reagent bead or microsphere. Each of the pockets, recesses orbores 21 provided in the base of the sample well 19 can also comprise acountersunk portion or region at the entrance to each tapered bore.According to an embodiment a single reagent bead or microsphere isdispensed and inserted into each pocket, recess or bore 21.

FIG. 2C shows in further detail a reagent bead or microsphere 20Adisposed and securely located in a pocket, recess or bore 21 provided inthe base of a sample well 19. The reagent bead or microsphere 20A issecured within the pocket, recess or bore 21. According to theembodiment shown in FIG. 2C the upper surface of the reagent bead ormicrosphere 20A when secured or located within the pocket, recess orbore 21 is positioned or located approximately 0.3 mm below the surfaceof the well bottom. Therefore, according to this embodiment reagentbeads or microspheres 20A located and secured in the pockets, recessesor bores 21 provided in the bottom of a sample well 19 do not projectabove the entrance to or surface of the pocket, recess or bore 21 andhence do not project above the bottom surface of the sample well 19.However, according to other embodiments one or more reagent beads ormicrospheres may be located in one or more pockets, recesses or bores 21provided in the base of the sample well 19 and may be located inrelatively shallow pockets, recesses or bores 21 or may be located inone or more pockets, recesses or bores 21 which have a taper such thatwhen the reagent bead or microsphere 20A is securely positioned withinthe pocket, recess or bore 21 then the reagent bead or microsphereprojects above the entrance into or surface of the pocket, recess orbore 21 and hence projects above the bottom surface of the sample well19. According to an embodiment reagent beads or microspheres 20A may bearranged such that they protrude 20-40% of their diameter above thebottom surface of the sample well.

Reagent beads or microspheres may be dispensed into pockets, recesses orbores 21 provided in the bottom of a sample well 19 of a sample plate bymeans of a reagent bead or microsphere dispenser 22 as will now bedescribed with reference to FIGS. 3A, 3B and 4. The loading ordispensing of reagent beads or microspheres may be performed either by asample plate manufacturer or by an end-user. A reagent bead ormicrosphere dispenser 22 is shown in FIG. 3A and can comprise an uppercap 23, a syringe body 24 and a barrel 25 which projects from a lowerregion of the syringe body 24.

FIG. 3B shows a cutaway view of the reagent bead or microspheredispenser 22 and shows that according to an embodiment the reagent beador microsphere dispenser further comprises a plunger guide 26 which ispositioned within the body of the syringe body 24. The plunger guide 26can comprise a screw thread on the outer surface of an upper portion ofthe plunger guide 26. The inner surface of an upper portion of thesyringe body 24 preferably comprises a complementary screw thread whichengages with the screw thread provided on the outer surface of the upperportion of the plunger guide 26 so that in use the plunger guide 26 issecured or screwed thinly to the syringe body 24. The inner surface ofthe cap 23 can also preferably comprise a screw thread and the cap 23also preferably screws onto the upper portion of the plunger guide 26.

A plunger 27 can be located within the plunger guide 26 and the plunger27 may be depressed by actuating an actuator or plunger boss 28 which islocated above the plunger 27 in the bore defined by the plunger guide26. An actuator spring (not shown) is provided between the actuator orplunger boss 28 so that when the actuator or plunger boss 28 isdepressed, force is transmitted to the plunger 27 via the actuatorspring causing the plunger 27 to become depressed. A return spring (notshown) can be provided between the bottom portion of the plunger guide26 and the plunger 27 so that when the actuator or plunger boss 28 is nolonger depressed, both the plunger 27 and the actuator or plunger boss28 are returned to an upper position.

FIG. 4 shows an exploded view of the reagent bead or microspheredispenser 22 as shown and described above with reference to FIGS. 3A and3B. FIG. 4 also shows that a silicone member 30 can be provided withinthe upper portion of the barrel 25. In use, reagent beads ormicrospheres within the syringe body 24 can be funneled or channeled bya helical path formed in the bottom section of the syringe body 24 sothat at the bottom of the syringe body 24 reagent beads or microspheresbecome arranged in single file or in series. The single file or seriesof reagent beads or microspheres leads into a chamber which can bearranged immediately above the barrel 25 and below the plunger guide 26.The chamber is shaped and arranged so as to accommodate a single reagentbead or microsphere which is positioned in a bore below the plunger 27and above the barrel 25. When the plunger 27 is depressed, the plunger27 can push a single reagent bead or microsphere 20A located in thechamber in a downwards direction. The single reagent bead or microsphere20A can forced by the plunger 27 through the silicone member 30.According to an embodiment the plunger 27 continues to push or urge thereagent bead or microsphere 20A through the barrel 25 and into a pocket,recess or bore 21 of a sample well 19 which can be positionedimmediately below the barrel 25 of the reagent bead or microspheredispenser 22. The silicone member 30 can prevent the accidental releaseof reagent beads or microspheres from the chamber of the reagent bead ormicrosphere dispenser 22 into the barrel 25 of the syringe body 24.

The bottom portion of the syringe body 24 can have a helical shape andact to guide or channel reagent beads or microspheres towards thechamber disposed in a lower portion of the syringe body 24. The chambercan be arranged so that only a single reagent bead or microsphere sitsabove the silicone member 30 at any instance in time. The chamber isformed in the bore through which the plunger 27 travels and depressionof the plunger 27 can cause a reagent bead or microsphere located in thechamber to be urged through the silicone member 30 and into the barrel25.

A vibration mechanism may optionally be provided and may be arranged toact on the outside of the syringe body 24 so as to ensure that reagentbeads or microspheres move down through syringe body 24 to the bottomportion of the syringe body 24 and line up in single file or in seriesready to enter the chamber.

Reagent beads or microspheres may be pre-packed or pre-loaded into thesyringe body 24 by, for example, a kit manufacturer or other supplier.Alternatively, an end-user may load the syringe body 24 with reagentbeads or microspheres. According to another embodiment the sample platemanufacturer may load the syringe body 24 with reagent beads ormicrospheres and may supply sample plates, sample strips or individualsample wells which are pre-loaded with one or more reagent beads ormicrospheres.

A microarrayer or automated apparatus will now be described withreference to FIG. 5. As shown in FIG. 5, a plurality of syringe bodies37 may be loaded onto a tray or pack 36 which can then be automaticallyloaded into the microarrayer or automated apparatus. The tray or pack 36comprising a plurality of syringe bodies 37 may be moved by a three-axistranslation mechanism or robotic arm to a reagent bead or microspheredispensing work area of the microarrayer or automated apparatus.

The microarrayer or automated apparatus can comprise a three-axistranslation mechanism which comprises a first translation stagecomprising a guide rail 31 along which a first arm 32 may be translatedin a first (x) horizontal direction. A second translation stage can beprovided and comprises a mounting block 33 which encompasses orsurrounds the first arm 32. The mounting block 33 may be translated in asecond (y) horizontal direction (which is preferably orthogonal to thefirst (x) horizontal direction) and may be moved backwards and forwardsalong the first arm 32. A third translation stage can be provided andcan comprise a body or syringe drive mechanism 34 which houses a linearactuator (not shown). The body or syringe drive mechanism 34 can beslidably mounted on the mounting block 33 and may be raised and loweredin a vertical (z) direction.

The three-axis translation mechanism can further comprise a retractablearm 35 which extends from the mounting block 33. The three-axistranslation mechanism can be programmed to select and pick up a reagentbead or microsphere dispenser 22,37 from the tray or pack 36 comprisinga plurality of reagent bead or microsphere dispensers 22,37. The body orsyringe drive mechanism 34 comprises a tapered spigot which isresiliently mounted within a tubular housing. The spigot is arranged toengage with a tapered portion provided on the syringe cap 23 of thereagent bead or microsphere dispenser 22,37. When a reagent bead ormicrosphere dispenser 22,37 is positioned in the tray or pack 36 thespigot may be lowered onto the syringe cap 23 of a reagent bead ormicrosphere dispenser 22,37 thereby securing the reagent bead ormicrosphere dispenser 22,37 to the body or syringe drive mechanism 34 ina detachable manner. The body or syringe drive mechanism 34 and attachedreagent bead or microsphere dispenser 22,37 may then be raised to aheight such that the retractable arm 35 (which is initially retractedwithin the body of the mounting block 33) can then be extended. Thereagent bead or microsphere dispenser 22,37 is then lowered by the bodyor syringe drive mechanism 34 so that the upper portion of the syringebody 24 is secured by the retractable arm 35. The retractable arm 35 canhave an aperture having an internal diameter which is preferably smallerthan the outermost diameter of a rim of the upper portion of the syringebody 24.

According to an embodiment each reagent bead or microsphere dispenser22,37 comprises a plurality of identical reagent beads or microspheres.According to an embodiment up to 15 separate reagent bead or microspheredispensers 22,37 may be loaded or provided in a single tray or pack 36and each of the reagent bead or microsphere dispensers 22,37 may have acapacity of up to approximately 2000 reagent beads or microspheres.

According to an embodiment the syringe drive mechanism 34 is arranged topick a reagent bead or microsphere dispenser 22,37 out of the tray orpack 36 and will position and lower the barrel 25 of the reagent bead ormicrosphere dispenser 22,37 so that it is immediately above a desiredreagent bead or microsphere pocket or recess 21 provided in a samplewell 19 of a sample plate. The syringe drive mechanism 34 is thenactuated so that the actuator or plunger boss 28 of the reagent bead ormicrosphere dispenser 22,37 is depressed which in turn causes theplunger 27 to push a reagent bead or microsphere 20A from the chamberthrough the silicone member 30, through the barrel 25 and into thedesired reagent bead or microsphere pocket or recess 21 of the samplewell 19. The syringe drive mechanism 34 can be arranged to depress theactuator boss 28 and plunger 27 with a desired amount of force asopposed to moving the actuator or plunger boss 28 and plunger 27 to acertain vertical position. As a result, reagent beads or microspheres20A are pressed in tightly and consistently into the reagent bead ormicrosphere pockets or recesses 21 of a sample well 19 with a constantamount of force.

FIG. 6 shows in greater detail a reagent bead or microsphere dispenserpick-up device or syringe drive mechanism 34 during the process ofpicking up a reagent bead or microsphere dispenser 22. The reagent beador microsphere dispenser pick-up device or syringe drive mechanism 34comprises a spigot 39 having a tapered lower end which is arranged toengage with a tapered recess provided in the upper portion of thesyringe cap 23 of the reagent bead or microsphere dispenser 22. Thespigot 39 comprises a central bore through which a plunger push rod 40is mounted. The plunger push rod 40 is arranged to be driven upwards ordownwards by a linear actuator 41 which drives a linear actuator leadscrew 42 which in turn raises or lowers the plunger push rod 40.

As shown in FIG. 6, in order to pick up a reagent bead or microspheredispenser 22 the reagent bead or microsphere dispenser pick-up device orsyringe drive mechanism 34 is lowered onto the reagent bead ormicrosphere dispenser 22 so that the spigot 39 of the reagent bead ormicrosphere pick-up device or syringe drive mechanism 34 engages withthe syringe cap 23 of the reagent bead or microsphere dispenser 22. Asthe reagent bead or microsphere dispenser pick-up device or syringedrive mechanism 34 is driven downwards onto the reagent bead ormicrosphere dispenser 22, the spigot 39 becomes compressed and movesupwards until it is prevented from moving any further upwards. Thespigot 39 can be driven further downwards whilst in a compressed stateso that the interlocking tapers of the spigot 39 and syringe cap 23engage causing the reagent bead or microsphere dispenser 22 to becomeattached to the reagent bead or microsphere pick-up device or syringedrive mechanism 34.

The reagent bead or microsphere dispenser 22 as shown in FIG. 6 issubstantially similar to that shown in FIGS. 3A, 3B and 4 except thatthe spacer 29 shown in FIGS. 3B and 4 is replaced with a retaining cap43 in the embodiment shown in FIG. 6. FIG. 6 also shows the location ofan actuating spring 44 which is provided between the actuator or plungerboss 28 and the plunger 27 and which transmits force applied to theactuator or plunger boss 28 to the plunger 27. A return spring 45 isalso shown and is provided between the plunger 27 and the base of theplunger guide 26 and causes the plunger 27 (and hence also the actuatoror plunger boss 28) to return to an upper position when the actuator orplunger boss 28 is no longer depressed or actuated.

FIG. 7A shows the reagent bead or microsphere dispenser pick-up deviceor syringe drive mechanism 34 which has picked up a reagent bead ormicrosphere dispenser 22 and which is in the process of transporting thereagent bead or microsphere dispenser 22 to a desired location. Once thereagent bead or microsphere dispenser pick-up device or syringe drivemechanism 34 has engaged with the reagent bead or microsphere dispenser22, the reagent bead or microsphere dispenser pick-up device or syringedrive mechanism 34 is raised so that the spigot 39 is no longercompressed. The spigot 39 returns to a downward position and the reagentbead or microsphere dispenser 22 including syringe body 24 is locked onto the spigot 39 by the tapers on the spigot 39 and syringe cap 23.

FIG. 7B shows a reagent bead or microsphere dispenser 22 in the processof dispensing a reagent bead or microsphere 20A from the reagent bead ormicrosphere dispenser 22 into a pocket or recess of a sample well (notshown) of a sample plate (not shown). The linear actuator 41 of thereagent bead or microsphere dispenser pick-up device or syringe drivemechanism 34 can be actuated and causes the linear actuator lead screw42 to extend thereby pushing the push rod 40 downwards. The downwardsmovement of the push rod 40 depresses the actuator or plunger boss 28.The actuator or plunger boss 28 transmits force to the plunger 27 viathe actuating spring 44 and in some embodiments, does not touch theplunger 27 directly. The plunger 27 forces a reagent bead or microsphere20A from a chamber within the central bore provided within the syringebody 24. The reagent bead or microsphere 20A can be forced through themembrane 30 and down through the barrel 25 and into the recess or pocketof a sample plate (not shown) by the plunger 27.

FIG. 8A shows the reagent bead or microsphere pick-up device or syringedrive mechanism 34 in the process of ejecting a reagent bead ormicrosphere dispenser 22 from the end of the reagent bead or microspherepick-up device or syringe drive mechanism 34. In this mode of operationthe reagent bead or microsphere dispenser 22 is positioned above thetray or pack 36. The linear actuator 41 drives the linear actuator leadscrew 42 downwards until the plunger 27 is extended a maximum extent.The spigot 39 is also extended to the maximum extent. The linearactuator 41 then continues to apply force via the actuator or plungerboss 28 to the plunger 27, as shown in FIG. 8B, with the result that thebody of the reagent bead or microsphere dispenser 22 can be forced offfrom the end of the tapered spigot 39. The reagent bead or microspheredispenser 22 then falls back into the reagent bead or microspheredispenser tray or pack 36.

In order to illustrate aspects of an embodiment of the present inventiona test was performed wherein a sample plate comprising nine sample wells19 was provided. Each sample well 19 comprised ten pockets, recesses orbores 21 which were arranged in a circle around a central portion of thesample well 19. Each of the pockets, recesses or bores 21 were loadedwith reagent beads or microspheres which were coated with differentconcentrations of reagent. The ten beads in the first sample well werecoated with a reagent having a concentration of 10 μg/ml and the tenbeads in the second sample well were coated with a reagent having aconcentration of 8 μg/ml. The ten beads in the third sample well werecoated with a reagent having a concentration of 4 μg/ml and the tenbeads in the fourth sample well were coated with a reagent having aconcentration of 2 μg/ml. The ten beads in the fifth sample well werecoated with a reagent having a concentration of 1 μg/ml and the tenbeads in the sixth sample well were coated with a reagent having aconcentration of 0.5 μg/ml. The ten beads in the seventh sample wellwere not coated with a reagent i.e. the concentration was 0 μg/ml. Theten beads in the eighth sample well were coated with differentconcentrations of reagent and comprised concentrations of 10 μg/ml, 8μg/ml, 4 μg/ml, 2 μg/ml, 1 μg/ml, 0.5 μg/ml, 0 μg/ml, 0 μg/ml, 0 μg/mland 0 μg/ml. The ten beads in the ninth sample well had the sameconcentrations as the reagent beads or microspheres in the eighth samplewell and were arranged in the same manner as the reagent beads ormicrospheres in the eighth sample well.

The reagent beads or microspheres were coated with a capture antibodycomprising sheep IgG and were transported in a bicarbonate buffercontaining 0.02% Kathon® preservative.

The sample wells 19 of the sample plate were emptied of the preservativein which the reagent beads or microspheres were transported in and 400μl of a 1/1000 diluted donkey anti-sheep IgG peroxidise conjugate in aTris Buffered Saline (“TBS”) conjugate diluent buffer was added to eachsample well 19. The sample plate was then incubated at ambienttemperature and was subjected to medium intensity vibrations for aperiod of 45 minutes. Any unbound conjugate was then aspirated from thesample wells 19 using a single channel wash head of a microarrayerapparatus (DS2®, available from Dynex Technologies). Once any unboundconjugate had been aspirated from the sample wells 19, 500 μl of 1/20diluted Tris Buffered Saline wash fluid was then immediately added toeach sample well 19. The wash fluid was then aspirated from the samplewells 19 and the process of washing and aspirating wash fluid from thesample wells 19 was repeated twice more. After the third washing stepincluding aspiration of wash fluid had been completed, 300 μl of luminol(a chemiluminescent marker) was then immediately added to each samplewell 19. The sample plate was then incubated in the dark at ambienttemperature whilst being subjected to medium intensity vibrations for 15minutes. The sample plate was then transferred immediately to a readingchamber.

A camera was set to an exposure time of 6 minutes and 30 seconds with again of 20. Images were taken at 22 minutes and 29 minutes after luminolhad been added. The camera exposure time was then changed to 8 minutesand 37 seconds. Further images were taken at 38 minutes, 47 minutes, 56minutes and 65 minutes after luminol addition. Analysis of the imagesshowed that the greatest observed signal strength was obtained after15-22 minutes from luminol addition which is consistent with the luminoldecay curve.

According to an embodiment the following steps may be carried out oncereagent beads or microspheres have been dispensed into pockets, recessesor bores of a sample plate. Firstly, sample fluid may be added to one ormore sample wells of the sample plate. The sample fluid may comprise oneor more analytes such as specific antigens which may react with reagentcoated on one or more of the reagent beads or microspheres. The reagentbeads or microspheres can be coated with a specific capture antibody.

Once the sample fluid has been added to the sample wells, the sampleplate can then be subjected to an incubation step. After the sampleplate has been subjected to an incubation step so that antigen-antibodycomplexes are formed, the sample plate can then be subjected to one ormore washing and aspirate steps in order to remove any unbound samplefluid and to remove any wash fluid. An enzyme conjugate can then addedwhich will bind to the antigen part of any antigen-antibody complexeswhich have been formed but which will not bind to antibodies or to theantibody part of an antigen-antibody complex. The sample plate can thenbe incubated before being subjected to one or more washing and aspiratesteps. Once the sample plate has been subjected to one or more washingand aspirate steps, luminol (or another visualizing agent) can be added.The sample plate is then aspirated to remove any excess luminol (orother visualizing agent). The luminol (or other visualizing agent) uponcontacting enzymes attached to the antigen part of an antigen-antibodycomplex can then breakdown causing a distinctive color to be produced.In the final stage the sample plate is analyzed and an endpointdetermination can be made.

An embodiment is shown in FIGS. 9A and 9B and will be described in moredetail below. FIG. 9A shows nine sample strips loaded into a plateframe. Each of the sample strips shown in FIG. 9A comprises a 6×1 stripof sample wells. The sample strips can be removeably loaded into theplate frame. Each of the nine sample strips comprises six sample wellsand each sample well can comprise ten (optionally tapered) bores which,in use, are arranged to receive a reagent bead. The reagent beads canthen be loaded or pre-loaded into the bores such that the reagent beadsprotrude above the base portion of the sample well. FIG. 9B shows theplate frame into which the sample plates may be loaded in more detail.

FIG. 10A shows in greater detail a sample strip comprising six samplewells. According to an embodiment the sample wells in a strip can beseparated or otherwise broken apart. According to an embodiment thesample plate or strip can be separated or divided up into single samplewells. FIG. 10B shows a sample strip comprising six sample wells beingloaded into a plate frame.

FIG. 11A shows a single sample well (which has been separated from astrip of sample wells) being loaded into a plate frame. The sample wellscan comprise a female portion which is arranged to engage or interlockwith a male portion which can be provided on the base of the plateframe. The sample plate or sample strip can be arranged to be firmlysecured and fixed to the plate frame when loaded onto the plate frame.

FIG. 11B shows in greater detail two sample wells which are connected bya break-apart feature 47. The break-apart feature 47 can allow a user toseparate adjacent sample wells. According to an embodiment sample wellsmay be separated from each other but may still be placed next to eachother on the plate frame without interfering with each other. Thebreak-apart feature 47 can comprise one, two or more than two breakpoints 46. According to an embodiment the connecting piece 47 betweentwo sample wells may be separated from a sample well at a first breakpoint 46. The connecting piece 47 may then be broken off or otherwiseremoved from the single sample well that it is attached to by breakingthe connecting piece 47 from the sample well at a second break point 46.

FIG. 11C shows a sample well having an end break-apart feature 48. Theend break-apart feature 48 can allow the end wells to be used singly inthe plate frame without interfering with another sample well. The endbreak-apart feature 48 provides something for a user to hold in order toremove a strip of sample wells or a single sample well from the plateframe.

FIG. 11D shows a sample well having an ID and orientation tab 49. Thetab 49 can allow an identifier to be printed onto the tab 49 or to beotherwise attached to the tab 49. The identifier may comprise a 2D or 3Dbarcode and/or human readable text. The tab 49 can assist a user toorientate a sample well when a single sample well is used by aligningwith features in the plate frame and/or on other sample wells.

FIG. 12A shows the underneath of a strip of sample wells and shows thataccording to an embodiment each sample well comprises ten bores orrecesses in which a reagent bead can be inserted in use. The base orunderside of each sample well can also comprise a female portion whichcan be arranged to be mated, in use, with a male portion which can beprovided in the base of the plate frame.

FIG. 12B shows in greater detail a female alignment and retainingfeature 50 which helps to align a strip of sample wells with a plateframe. FIG. 12C shows a corresponding male alignment and retainingfeature 51 which can be provided in the base of the plate frame. Themale portion 51 may according to an embodiment comprise a plurality offlexible projections which are preferably deformed inwards as a samplewell is located over the male portion 51. The projections on the plateframe can move or close together ensuring that the sample well is keptin place without having to apply undue force either to mount or fix asample well onto the plate frame and/or to demount a sample well fromthe plate frame.

FIG. 13 shows a cross-sectional view of a strip of sample wells andshows that according to an embodiment the sample wells may comprise aplurality of tapered bores 52. The tapered bores 52 preferably act aspockets into which a reagent bead is inserted in use. The angle of thetaper in the embodiment shown in FIG. 13 is 6.0°.

Although various embodiments described above have focused upon reagentbeads which are coated with a biomolecule for use in an Immunoassay orELISA procedure, other embodiments equally apply to reagent beads whichcomprise or which are otherwise coated with a nucleic acid sequence andwhich are used as a hybridization probe for the detection of DNA or RNAsequences which are complementary to those provided on the reagentbeads. In some embodiments, the hybridization probe will be inactiveuntil hybridization, at which point there is a conformational change andthe molecule complex becomes active and will then fluoresce under UVlight. Therefore, all the various embodiments described above and allthe various aspects of the embodiments described above apply equally tothe use of reagent beads comprising or which are otherwise coated with aDNA or RNA sequence (or other nucleotide sequence) for use as ahybridization probe to detect complementary DNA or RNA sequences.

Many variants, including fluorogenic and luminogenic substrates forELISA, direct labeling of the second member of the binding pair with afluorescent or luminescent molecule (in which case the procedure is notcalled an ELISA but the process steps are very similar) and nucleicacids or other specific pairing agents instead of antibodies can be usedas a probe. The same principles can be used to detect or determine anymaterials which can form specific binding pairs, for example usinglectins, rheumatoid factor, protein A or nucleic acids as one of thebinding partners.

The sample plate can thus be used to detect an analyte, such as abiomarker, which can be indicative of a disease or condition. Thedisease or condition can be a tumor, neoplasm, or cancer, such as breastcancer, ovarian cancer, lung cancer, colon cancer, hyperplastic polyp,adenoma, colorectal cancer, high grade dysplasia, low grade dysplasia,prostatic hyperplasia, prostate cancer, melanoma, pancreatic cancer,brain cancer (such as a glioblastoma), hematological malignancy,hepatocellular carcinoma, cervical cancer, endometrial cancer, head andneck cancer, esophageal cancer, gastrointestinal stromal tumor (GIST),renal cell carcinoma (RCC) or gastric cancer. The disease or conditioncan also be an inflammatory disease, immune disease, or autoimmunedisease, such as inflammatory bowel disease (IBD), Crohn's disease (CD),ulcerative colitis (UC), pelvic inflammation, vasculitis, psoriasis,diabetes, autoimmune hepatitis, Multiple Sclerosis, Myasthenia Gravis,Type I diabetes, Rheumatoid Arthritis, Psoriasis, Systemic LupusErythematosis (SLE), Hashimoto's Thyroiditis, Grave's disease,Ankylosing Spondylitis Sjogrens Disease, CREST syndrome, Scleroderma,Rheumatic Disease, organ rejection, Primary Sclerosing Cholangitis, orsepsis. The disease or condition can also be a cardiovascular disease,such as atherosclerosis, congestive heart failure, vulnerable plaque,stroke, ischemia, high blood pressure, stenosis, vessel occlusion or athrombotic event. The disease or condition can also be a neurologicaldisease, such as Multiple Sclerosis (MS), Parkinson's Disease (PD),Alzheimer's Disease (AD), schizophrenia, bipolar disorder, depression,autism, Prion Disease, Pick's disease, dementia, Huntington disease(HD), Down's syndrome, cerebrovascular disease, Rasmussen'sencephalitis, viral meningitis, neurospsychiatric systemic lupuserythematosus (NPSLE), amyotrophic lateral sclerosis, Creutzfeldt-Jacobdisease, Gerstmann-Straussler-Scheinker disease, transmissiblespongiform encephalopathy, ischemic reperfusion damage (e.g. stroke),brain trauma, microbial infection, or chronic fatigue syndrome. Thephenotype may also be a condition such as fibromyalgia, chronicneuropathic pain, or peripheral neuropathic pain. The disease orcondition can also be an infectious disease, such as a bacterial, viralor yeast infection. For example, the disease or condition may beWhipple's Disease, Prion Disease, cirrhosis, methicillin-resistantstaphylococcus aureus, HIV, hepatitis, syphilis, meningitis, malaria,tuberculosis, or influenza. Viral proteins, such as HIV or HCV-likeparticles can be assessed in an exosome, to characterize a viralcondition.

The sample plate can be used to detect a biomarker that is used todetect the disease or condition. For example, the detection of abiomarker can be used to detect or provide a diagnosis, prognosis of adisease or condition. For example, the sample plate can comprise a probefor a cancer marker, and used to detect the cancer marker in a samplefrom an individual. The presence, absence, or level of the cancer markerin the sample can be indicative of cancer in the individual. In anotherembodiment, the sample plate can also be used to monitor a disease orcondition. For example, an increased level of the cancer marker, ascompared to a control, or compared to an earlier assay for the cancermarker from the same individual, can be indicative of progression of thecancer. In yet another embodiment, the sample plate can be used to indetermine a therapy or course of action for a condition. For example, anindividual may have a genetic variant which leads to the individualbeing unable to metabolize certain drugs. The sample plate can be usedto detect the genetic variant. In another embodiment, the sample platemay be used to detect a compound, which can be indicative of a drug notbeing metabolized. The sample plate can also be used to detect theintake of certain drugs or compounds, such as be detecting the drug orby-products of the drug, which can be used for drug testing.

The sample plate can also be used to screen for drugs. For example, thesample plate can comprise a probe that is a target for drug development.The sample plate can then be used to screen a library of compounds.Alternatively, the sample plate can comprise a plurality of probes thatcomprise a library of compounds that are potential drugs. The sample cancomprise a drug target, which is added to the sample plate.

Also provided herein is a kit comprising a sample plate disclosedherein. The kit can comprise one or more components for detecting ananalyte or for performing an assay. In one embodiment, a kit fordetecting an analyte comprises one or more sample plates and a pluralityof beads. The plurality of beads can comprise one or more probes, suchas a probe that is a nucleic acid, antibody, antibody fragment, protein,peptide, aptamer, or chemical compound. In another embodiment, a kit forperforming an Enzyme Linked Immunosorbent Assay (ELISA) procedure isprovided. The kit can comprise one or more sample plates as describedherein; and a plurality of beads, wherein the beads are coated with areagent comprising an antibody, an antigen or another biomolecule. Inyet another embodiment, the kit can comprise components for performing anucleic acid probe procedure, wherein the kit comprises one or moresample plates as described herein; and a plurality of beads coated witha nucleic acid, such as a DNA or RNA probe or sequence.

Further embodiments of the present invention will now be described withreference to FIGS. 14A and 14B. According to the embodiment shown inFIG. 14A reagent beads 53 are loaded into a sample plate from theunderneath or rear side of the sample plate. The sample plate comprisesa bore or through hole 54 which according to the embodiment as shown inFIG. 14A is tapered. However, as will be discussed below, it is alsocontemplated that the bore or through hole may not be tapered and mayinstead comprise a substantially cylindrical through hole or bore 54which has a substantially constant cross-sectional diameter and/or areaand/or profile. FIG. 14B shows a sample plate according to an embodimentof the present invention wherein reagent beads or microspheres aresecured within a cylindrical bore or through hole 54. The reagent beadsor microspheres may be inserted into the cylindrical bore or throughhole 54 either from the top or from the bottom. The reagent beads ormicrospheres can be secured within the bore or through hole 54 by aninterference fit and the reagent beads or microspheres make asubstantially fluid-tight seal around a full circumference of, perimeterof or closed loop around the reagent bead or microsphere.

With regard to the embodiment shown in FIG. 15 and referring back toFIG. 14A, bores or through holes 54 in a sample well may taper from afirst diameter at the lowermost part or bottom of the base portion 55 ofthe sample well 56 to a second narrower diameter towards the uppermostpart or top of the base portion 55. The uppermost part or top of thebase portion 55 is that part of the base portion 55 which comes intocontact with sample fluid in use.

At the top of the bore or through hole 54 immediately below the portionof the base portion 55 which comes into contact with sample fluid, thebore or through hole 54 may be shaped so as to form a tight fit with areagent bead 53. The uppermost portion of the bore or through hole maycomprise a part spherical profile, bulbous region, curved portion orconcave region so that a reagent bead 53 which is inserted into the boreor through hole 54 from the underneath of the sample plate fits tightlywithin the part spherical profile, bulbous region, curved portion orconcave region at the top of the bore or through hole 54 as shown inFIG. 14A.

According to an embodiment at least a portion of the reagent bead 53 isarranged to project into the base or bottom of the sample well to form,in effect, part of the base portion of the sample well 56. As a result,the top portion of the reagent bead 53 (above the region where the beadforms a fluid-tight circumferential seal with the wall of the throughhole) is arranged so as to come into contact with sample fluid in use.The reagent bead 53 forms a fluid tight seal around the fullcircumference of the bead 53 with the part spherical profile, bulbousregion, curved portion or concave region of the bore or through hole 54.

According to an embodiment macro sized beads 53 are fitted into a samplewell 56 of a sample plate so that only the top or upper portion of thereagent bead 53 is exposed to fluid. It should be noted that theluminescent reading process is a 2D operation and only takes intoaccount signal from the visible portion of the reagent bead 53 facingthe camera.

According to an embodiment the multiplex well together with reagentbeads loaded into the through holes preferably mimics the wellestablished microplate ELISA type of process. The multiplex wellaccording to an embodiment is substantially similar in format to amicroplate well.

One of the major factors in processing an ELISA test in a microplate isthe efficiency or cleanliness of each step. Any residual fluid from thesteps can have an overall effect on the performance of the test e.g. ifthe conjugate is not completely removed by washing, then residualconjugate will produce a false signal on the bead. This will drive downthe sensitivity of the test by increasing the background signal.

One aspect of efficient processing of the test is not to have any fluidtraps in the well. Any corners, pockets or undercuts may trap fluidthereby reducing the performance of the sample plate. The sample plateaccording to an embodiment can allow efficient washing, mixing andaspirating in a similar manner to a conventional microplate well and insome embodiments does not suffer from the problem of trapping fluid.

In some embodiments beads 53 can be fitted at a uniform height in asample well 56 which can ensure that each bead 53 is treatedidentically. Each bead 53 makes a fluid tight sealed fit in the locatingdetail of a pocket of through hole to ensure that there is no fluidtrapped under or below the bead 53.

The through hole 54 may comprise a tapered conical hole in which thebead locks into the hole as shown in FIG. 14A or the through hole 54 maycomprise a cylindrical undersized hole into which a bead is mechanicallypressed into as shown in FIG. 14B. Both embodiments can achieve the goalof preventing fluid going past the bead 53 and becoming trappedunderneath or below the bead 53.

If the sample plate comprises one or more tapered through holes 54 asshown in FIG. 14A then the through holes can be manufactured with a highdegree of accuracy and consistency to ensure that beads are securedwithin the sample plate at a uniform height (since the reagent beads 53can be pressed into the through holes 54 with a set force and not to aset height). The alternative embodiment of using undersized cylindricalthrough holes as shown in FIG. 14B may not need to be manufactured to sosuch a high degree of accuracy since the reagent beads 53 can be pressedin to the through holes to a set height and not with a set force.

In some of the embodiments described above reagent beads may be fittedinto a blind pocket detail in a sample well i.e. into a closed recess.However, in some embodiments, a sample plate having through holes in thebase portion may be provided as shown and described above with referenceto FIGS. 14A and 14B.

The assembly of a sample plate which is loaded with reagent beads duringproduction or manufacture can be subjected to a quality control check toensure that all the beads are sealed to the sample plate. Beads whichare loaded into blind pockets as described above can ensure that fluidwill not leak out of the well. However, fluid might still leak under thebead and such a leak would be difficult to detect.

According to an embodiment, a sample plate comprising through holes asshown in FIGS. 14A and 14B can allow a pressure check to be carried outas part of the bead to plate assembly, manufacture and quality controlchecks. This can ensure that the bead to plate seal is good. A defectivebead or damaged hole would show up as a fail in the manufacture and notwhen the user runs the test.

The sample plate according to the embodiments as shown in FIG. 14A oroptionally also in FIG. 14B wherein reagent beads are fitted into thebore from underneath can be particularly advantageous for a number ofreasons. Firstly, contact between a press in tool and the bead 53 iswith the bottom or underneath portion of the reagent bead 53 so anywitness mark can also be on the bottom or underneath portion of thereagent bead 53 i.e. not any portion of the reagent bead 53 which willcome into contact with sample fluid. Secondly, the top of the throughhole 53 in the base portion 55 of the sample well in the example shownin FIG. 14A can be made to match the profile or shape of the reagentbead 53 so that no moat portion is formed around the portion of the bead53 which protrudes into the base of the sample plate. As a result, thedesign can minimize or exclude any possibility of trapping fluid belowthe reagent bead 53. Thirdly, it can minimize or eliminate crosscontamination between beads since the press in tool will only come intocontact with the underneath or bottom portion of the reagent beads53—the press in tool does not come into contact with the top portion ofthe reagent beads 53 (i.e. the portion of the reagent beads 53 whichwill come into contact with sample fluid). Fourthly, in the embodimentshown in FIG. 14A reagent beads 53 can be fitted lower in the baseportion without forming a moat region and in a manner which reduces therisk of crosstalk.

According to an embodiment fluid is only arranged to come into contactwith the top surface of a reagent bead 53. According to an embodimentfluid is prevented from passing down a through hole 54 or recess past areagent bead 53 secured within the through hole 54 or recess.

A sample plate according to an embodiment can be cleaned easily duringthe process steps without trapping fluid under the reagent beads 53. Thebeads 53 can be provided in a format that makes it as close to acylindrical well as possible and which can also be easily accessed fromthe top.

FIG. 15 shows an embodiment wherein a strip of six sample wells withfive 3 mm reagent beads loaded into through holes in each sample well.The reagent beads can be loaded into the through holes from the bottomor underneath of the sample plate. The reagent beads can be retainedwithin the through holes by upper concave regions formed in the throughholes.

FIG. 16 shows a three dimensional cross-sectional view of thearrangement as shown and described above with reference to FIGS. 14A and15.

FIG. 17 shows a further embodiment of the present invention wherein thebase portion of a sample well is sub-divided into a plurality ofsegments 57A-E. According to an embodiment each base portion segment57A-E has one or more open through holes provided in the base portionsegment so that a reagent bead can be inserted from above or below intothe open through hole. According to another embodiment each base portionsegment 57A-E may have one or more blind recesses provided in the baseportion segment so that a reagent bead can be inserted from above intothe blind recess. According to another embodiment some of the baseportion segments 57A-E may comprise one or more through holes and otherbase portion segments 57A-E may comprise one or more blind recesses.According to an embodiment some or all of the through holes and/orrecesses are non-tapered and comprise a cylindrical bore. However,according to another embodiment some or all of the through holes and/orsome or all of the recesses may be tapered.

According to an embodiment reagent beads or microspheres are retained orsecured, in use, within the through holes and/or recesses provided inthe base portion segments so as to form a substantially fluid-tightcircumferential seal with a wall of the base portion segment whichdefines the through hole and/or the recess.

The base portion segments 57A-E may be arranged in a spiral or otherstaggered arrangement in a similar manner to that shown in FIG. 17. Thebase portion segments 57A-E can be arranged at different relativeheights to each another so that once reagent beads have been insertedinto the open through holes or recesses provided in the base portionsegments 57A-E then there is no direct line of sight between adjacentreagent beads (or any line of sight between adjacent reagent beads issignificantly reduced). In the embodiment shown in FIG. 17, base portionsegment 57A is relatively higher than base portion segment 57B; baseportion segment 57B is relatively higher than base portion segment 57C;base portion segment 57C is relatively higher than base portion segment57D; and base portion segment 57D is relatively higher than base portionsegment 57E. Embodiments wherein there is no (or alternatively, areduced) direct line of sight between reagent beads inserted intothrough holes and/or recesses in base portion segments provided withinthe same sample well (including the embodiment shown and described abovewith reference to FIG. 17) can reduce or eliminate crosstalk betweenreagent beads when the reagent beads are subsequently optically analyzedto determine the intensity of a reaction. According to an embodiment thereagent beads include an indicator which during an analysis step isilluminated by a light source and the intensity of the indicator on areagent bead is determined by a detector such as a camera to give ameasure of the intensity of a reaction.

FIGS. 18A and 18B show another embodiment wherein a low-height baffle 58is provided in a sample well so as to sub-divide the base portion of thesample well into a first base portion 59 having two open through holesor recesses and a second base portion 60 having three open through holesor recesses. It will be understood that other embodiments arecontemplated wherein the first base portion 59 and/or the second baseportion 60 may comprise a greater or lesser number of open through holesor recesses.

FIGS. 19A and 19B show another embodiment wherein a low-height baffle 61is provided in the sample well. According to the particular embodimentshown in FIGS. 19A and 19B, the baffle 61 sub-divides the base portionof the sample well into a first base portion 62A having four openthrough holes or recesses and a second base portion 62B also having fouropen through holes or recesses.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A sample plate comprising: at least one samplewell including: a base portion; a plurality of recesses provided in saidbase portion; and a plurality of beads received in said plurality ofrecesses, wherein each of said plurality of beads is retained or securedwithin a respective one of said plurality of recesses to substantiallyfluid-tightly seal the base portion at said plurality of recesses. 2.The sample plate of claim 1, wherein each of the plurality of recessesis defined, at least in part, by a respective wall of said base portionand each of said plurality of beads forms a substantially fluid-tightcircumferential seal around the respective wall of said base portion. 3.The sample plate of claim 1, wherein said plurality of recessesconstitute blind recesses formed in said base portion.
 4. The sampleplate of claim 1, wherein said plurality of recesses constitute openthrough holes formed in said base portion.
 5. The sample plate of claim1, wherein each of said plurality of recesses is substantiallycylindrical.
 6. The sample plate of claim 1, wherein each of saidplurality of recesses is conical and has a first diameter which isgreater than a diameter of a respective one of said plurality of beadsdeposited in said each of said plurality of recesses and a seconddiameter which is less than the diameter of said respective one of saidplurality of beads.
 7. The sample plate of claim 1, wherein saidplurality of recesses are arranged circumferentially around a centralportion of said sample well.
 8. The sample plate of claim 1, whereinsaid base portion is segmented into a plurality of segments which arearranged at different heights relative to each other.
 9. A sample platecomprising: at least one sample well including: a base portion, whereinsaid base portion is segmented into a plurality of segments which arearranged at different heights relative to each other; a plurality ofrecesses provided in said base portion; and a plurality of beadsreceived in said plurality of recesses.
 10. The sample plate of claim 1,wherein said at least one sample well further comprises one or morebaffles or dividers which separates or divides said base portion into atleast a first region and a second region.
 11. The sample plate of claim10, wherein said one or more baffles or dividers attenuate or eliminatelight reflected off certain ones of said plurality of beads which arelocated in said first region from impinging upon other ones of saidplurality reagent beads located in said second region.
 12. The sampleplate of claim 1, wherein at least some of said plurality of recessescomprise a countersunk portion.
 13. A sample well comprising: a baseportion; a plurality of recesses provided in said base portion; and aplurality of beads received in said plurality of recesses, wherein eachof said plurality of beads is retained or secured within a respectiveone of said plurality of recesses to substantially fluid-tightly sealthe base portion at said plurality of recesses.
 14. The sample well ofclaim 13, wherein each of the plurality of recesses is defined, at leastin part, by a respective wall of said base portion and each of saidplurality of beads forms a substantially fluid-tight circumferentialseal around the respective wall of said base portion.
 15. The samplewell of claim 13, wherein said plurality of recesses constitute blindrecesses formed in said base portion.
 16. The sample well of claim 13,wherein said plurality of recesses constitute open through holes formedin said base portion.
 17. The sample well of claim 13, wherein each ofsaid plurality of recesses is substantially cylindrical.
 18. The samplewell of claim 13, wherein each of said plurality of recesses is conicaland has a first diameter which is greater than a diameter of arespective one of said plurality of beads deposited in said each of saidplurality of recesses and a second diameter which is less than thediameter of said respective one of said plurality of beads.
 19. Thesample well of claim 13, wherein said plurality of recesses are arrangedcircumferentially around a central portion of said sample well.
 20. Thesample well of claim 13, wherein said base portion is segmented into aplurality of segments which are arranged at different heights relativeto each other.